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Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications
Abstract
Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) promote the proliferation of multiple myeloma (MM) cells and protect them against dexamethasone (Dex)-induced apoptosis. We have previously shown that Apo2 ligand/TNF-Related apoptosis inducing ligand (Apo2L/TRAIL) induces apoptosis of MM cells, including cells either sensitive or resistant to Dex and cytotoxic drugs, and overcomes the growth and survival effect of IL-6; conversely, NF-ΚB transcriptional activity attenuates their Apo2L/TRAIL-sensitivity. In the current study, we demonstrate that IGF-1 stimulates sustained activation of NF-ΚB and Akt; induces phosphorylation of the FKHRL-1 Forkhead transcription factor; upregulates a series of intracellular anti-apoptotic proteins including FLIP, survivin, cIAP-2, A1/Bfl-1, and XIAP; and decreases Apo2L/TRAIL-sensitivity of MM cells. In contrast, IL-6 does not cause sustained NF-ΚB activation, induces less pronounced Akt activation and FKHRL-1 phosphorylation than IGF-1, and increases the expression of only survivin. Forced overexpression of constitutively active Akt in MM-1S cells reduced their sensitivity to Apo2L/TRAIL and to doxorubicin (Doxo). In contrast, the Akt inhibitor IL-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate induced cell death of both Dex- and Doxo-sensitive and -resistant cells; opposed the protective effect of constitutive Akt activity against Apo2L/TRAIL; and abrogated the NF-ΚB activation, increase of anti-apoptotic proteins and protection against Apo2L/TRAIL induced by IGF-1. These findings therefore define an important role of the Akt pathway in modulating tumor cell responsiveness to Apo2L/TRAIL, delineate molecular mechanisms for the survival effects of IGF-1, and characterize differential pathophysiologic sequelae of IGF-1 vs IL-6 on MM cells. Importantly, they provide the basis for future clinical trials in MM combining conventional or novel agents with strategies designed to neutralize IGF-1.
... Overexpressing SNARE proteins involved in endoplasmic reticulum (ER) to Golgi transport overcome the vesicular arrest between the ER and Golgi caused by α-syn (Cooper et al., 2006;Gitler et al., 2008). Moreover, using this model, we and others found that α-syn leads to a pathological increase in cytosolic Ca 2+ , resulting in cell death (Chan et al., 2007;Danzer et al., 2007;Dufty et al., 2007;Guzman et al., 2010;Surmeier et al., 2010;Goldberg et al., 2012;Martin et al., 2012;Buttner et al., 2013;Hurley et al., 2013;Yuan et al., 2013;Caraveo et al., 2014;Surmeier et al., 2016;Caraveo et al., 2017;Surmeier et al., 2017;Betzer et al., 2018;Shum et al., 2023). Some data suggest that the source of this pathological elevation could be either Ca 2+ uptake at the plasma membrane via the L-type voltage-gated Ca 2+ channel Cav1.3 (Brown et al., 2006;Chan et al., 2007;Ilijic et al., 2011;Hurley and Dexter, 2012;Van Maele-Fabry et al., 2012;Dryanovski et al., 2013;Cali et al., 2014;Goldman, 2014;Schapira, 2015;Ortner and Striessnig, 2016;Surmeier et al., 2016;Zamponi, 2016) or α-syn itself, as its oligomeric conformation has the ability to form pores at the plasma membrane (Danzer et al., 2007;Angelova et al., 2016;Di Scala et al., 2016). ...
... An essential transducer of Ca 2+ gradients into cellular responses is the highly evolutionarily conserved Ca 2+ -calmodulin-dependent serine/threonine phosphatase, calcineurin (Aramburu et al., 2004). Our group and others have established a central role of calcineurin activity in α-syn pathobiology (Dufty et al., 2007;Guzman et al., 2010;Surmeier et al., 2010;Goldberg et al., 2012;Martin et al., 2012;Hurley et al., 2013;Caraveo et al., 2014;Surmeier et al., 2016;Burbulla et al., 2017). Using a range of model organisms, starting from yeasts and worms to primary cortical and dopaminergic neurons as well as in vivo rodent models of α-syn pathobiology, we found that diseaseassociated forms of α-syn lead to a pathological increase in cytosolic Ca 2+ and heightened calcineurin activity, culminating in neuronal death Surmeier et al., 2010;Goldberg et al., 2012;Hurley et al., 2013;Caraveo et al., 2014;Surmeier et al., 2016). ...
... The increased peripheral IGF-1 levels in α-syn transgenic mice treated with FK506 suggest a brain origin, as α-syn is not overexpressed in the periphery. IGF-1 expression can be regulated by calcineurin via NFAT (Alfieri et al., 2007) or by NF-κB via IkB kinase, a substrate for calcineurin (Mitsiades et al., 2002). Although IGF-1 can be produced by various cell types in the body, in CNS, neurons and glial cells, mainly astrocytes, serve as the main sources (Labandeira- Garcia et al., 2017;Shum et al., 2023). ...
Introduction: Rise in Calcium (Ca²⁺) and hyperactive Ca²⁺-dependent phosphatase calcineurin represent two key determinants of a-synuclein (a-syn) pathobiology implicated in Parkinson’s Disease (PD) and other neurodegenerative diseases. Calcineurin activity can be inhibited with FK506, a Food and Drug Administration (FDA)-approved compound. Our previous work demonstrated a protective effect of low doses of FK506 against a-syn pathology in various models of a-syn related pathobiology.
Methods: Control and a-syn-expressing mice (12-18 months old) were injected with vehicle or two single doses of FK506 administered 4 days apart. Cerebral cortex and serum from these mice were collected and assayed using a meso scale discovery quickplex SQ 120 for cytokines and Enzyme-linked immunosorbent assay for IGF-1.
Results: In this study we present evidence that reducing calcineurin activity with FK506 in a-syn transgenic mice increased insulin growth factor (IGF-1), while simultaneously decreasing IL-2 levels in both cerebral cortex and serum.
Discussion: The highly conserved Ca²⁺/calcineurin signaling pathway is known to be affected in a-syn-dependent human disease. FK506, an already approved drug for other uses, exhibits high brain penetrance and a proven safety profile. IL-2 and IGF-1 are produced throughout life and can be measured using standard clinical methods. Our findings provide two potential biomarkers that could guide a clinical trial of FK506 in PD patients, without posing significant logistical or regulatory challenges.
... There is clearly a need for specific PKB inhibitors to determine the role that PKB plays in vivo and to assess PKB as a potential chemotherapeutic target. Although many attempts towards the design of a PKB inhibitor have been made (Reuveni et al., 2002), a PKB inhibitor has only recently become available and used in cells (Mitsiades et al., 2002, Kozikowski, et al., 2003. ...
... Recently a PKB inhibitor has been designed and used in cells to inhibit the activation of all three PKB isoforms (Mitsiades et al., 2002). The PKB inhibitor, lL-6-Hydroxymethy 1-chiro-inosito 1 2-(R)-2-0-methyl-3-0-octadecylcarbonate, induced cell death in multiple myeloma cells that were both resistant to dexamethasone or doxorubicin (Mitsiades et al., 2002). ...
... Recently a PKB inhibitor has been designed and used in cells to inhibit the activation of all three PKB isoforms (Mitsiades et al., 2002). The PKB inhibitor, lL-6-Hydroxymethy 1-chiro-inosito 1 2-(R)-2-0-methyl-3-0-octadecylcarbonate, induced cell death in multiple myeloma cells that were both resistant to dexamethasone or doxorubicin (Mitsiades et al., 2002). Similarly, a novel phosphoinositide analogue that inhibits PKB is a potent inducer of apoptosis (Kozikowski et al., 2003). ...
p>Protein Kinase B (PKB), also known as Akt, was discovered in 1991 as the mammalian homologue of the viral oncogene product, v-akt. Subsequently, overexpression or constitutive activation of PKBα, PKBβ and PKBγ has been demonstrated in a number of cancers. The idea that inhibition of PKB in cancer cells may restore the sensitivity of cells to chemotherapeutic drugs has fuelled the search for specific inhibitors of PKB.
The aim was to manipulate PKB levels by overexpression or antisense technology to determine whether PKB regulates apoptosis. To test whether various PKB isoforms have the potential to regulate apoptosis in cells PKB levels were first modulated by overexpression. For this the activation of caspase-3 by staurosporine, an established induced or apoptosis, was employed. Overexpression of PKBα or PKBγ protected staurosporine-treated PC12 cells against the activation of caspase-3. To rigorously establish that these results were not non-specific effects of overexpression, antisense knockout of endogeneou PKB was then used to remove individual or combinations of PKB isoforms from cells. The combined depletion of greater than 95% PKBα, PKBβ and PKBγ from 3T3-L1 adipocytes sensitised cells to staurosporine-induced caspase-3 activation. Depletion of over 95% PKBα and PKBβ, or 95% of PKBγ alone, was not sufficient to sensitise 3T3-L1 adipocytes to the activation of caspase-3 induced by staurosporine. As staurosporine inhibited the activation of PKB, this effect requires PKB proteins and clearly indicates crucial roles of PKB in interacting with other proteins.
In conclusion these results show that endogenous PKB plays a critical role in regulating apoptosis as measured by caspase-3 activation. This antiapoptotic potential makes PKB an attractive drug target in cancer treatment.</p
... Another growth factor worth mentioning, which is known to serve as a proliferative and anti-apoptotic factor in MM, is IGF-1 (insulin-like growth factor 1) [128][129][130]. Interestingly, on the other hand, evidence exists that IGF-1 promotes migration by acting as a chemoattractant and impacts invasion in MM [131][132][133]. ...
... The migratory properties of IGF-1 are upregulated and correlated with epithelial-mesenchymal transition (EMT) markers in MM patients, which provides proof of the potential use of this growth factor in MM treatment [134]. It has been confirmed that IGF-1 induces activation of NF-B in MM cells and reduces the Apo2L/TRAIL sensitivity of these cells [128]. Interestingly, IGF-1 is mainly produced by the bone microenvironment, but is also present in the peripheral blood serum of MM patients at significantly increased levels [128]. ...
... It has been confirmed that IGF-1 induces activation of NF-B in MM cells and reduces the Apo2L/TRAIL sensitivity of these cells [128]. Interestingly, IGF-1 is mainly produced by the bone microenvironment, but is also present in the peripheral blood serum of MM patients at significantly increased levels [128]. The anti-apoptotic effect obtained by the IGF-1 differs from that achieved by IL-6 in MM individuals [128]. ...
Multiple myeloma (MM) is a plasma cell neoplasm characterized by an abnormal proliferation of clonal, terminally differentiated B lymphocytes. Current approaches for the treatment of MM focus on developing new diagnostic techniques; however, the search for prognostic markers is also crucial. This enables the classification of patients into risk groups and, thus, the selection of the most optimal treatment method. Particular attention should be paid to the possible use of immune factors, as the immune system plays a key role in the formation and course of MM. In this review, we focus on characterizing the components of the immune system that are of prognostic value in MM patients, in order to facilitate the development of new diagnostic and therapeutic directions.
... In the BMM, MM plasma cells are activated by several factors such as IL-6, JAK/STAT, rat sarcoma/mitogen activated protein kinase (Ras/MAPK), phosphatidylinositol-3 kinase (PI3-K)/Akt, and TNF family including B cell activating factor (BAFF), and a proliferation inducing ligand (APRIL). Additionally, IGF-1 can play a role in the survival of MM cells, which can activate NF-κB and Akt, as well as increase expression of FLIP and cIAP-2 which inhibit caspase-8 [114]. IGF-1 can also downregulate the expression of Bim, resulting in less antagonism of anti-apoptotic proteins [115]. ...
... inducing ligand (APRIL). Additionally, IGF-1 can play a role in the survival of MM cells, which can activate NF-κB and Akt, as well as increase expression of FLIP and cIAP-2 which inhibit caspase-8 [114]. IGF-1 can also downregulate the expression of Bim, resulting in less antagonism of anti-apoptotic proteins [115]. ...
Over the past two decades, the natural history of multiple myeloma (MM) has evolved dramatically, owing primarily to novel agents targeting MM in the bone marrow microenvironment (BMM) pathways. However, the mechanisms of resistance acquisition remain a mystery and are poorly understood. Autophagy and apoptosis are tightly controlled processes and play a critical role in the cell growth, development, and survival of MM. Genetic instability and abnormalities are two hallmarks of MM. During MM progression, plasma malignant cells become genetically unstable and activate various signaling pathways, resulting in the overexpression of abnormal proteins that disrupt autophagy and apoptosis biological processes. Thus, achieving a better understanding of the autophagy and apoptosis processes and the proteins that crosslinked both pathways, could provide new insights for the MM treatment and improve the development of novel therapeutic strategies to overcome resistance. This review presents a sufficient overview of the roles of autophagy and apoptosis and how they crosslink and control MM progression and drug resistance. Potential combination targeting of both pathways for improving outcomes in MM patients also has been addressed.
... More specifically, components of the Ras-MEK-ERK pathway (Ras, Raf, ERK, RSK) positively regulate PI3K-mTORC1 pathway [82]. We have also shown that Akt inhibitor IL-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate induces MM cell death associated with downregulation of the NF-κB activity, suggesting crosstalk between PI3K-Akt and NF-κB signaling pathways [85]. It also modulates cell cycle and proliferation both directly via activity on the CDK inhibitors p21 WAF1/Cip1 and p27 Kip1 , as well as indirectly by affecting the levels of p53 and cyclinD1. ...
... IκBα therefore has a crucial role in regulating canonical NF-κB activation. For example, various growth and/or anti-apoptosis promoting cytokines, including tumor necrosis factor (TNF)α [61,101,102] and IGF-1 [85], trigger phosphorylation of IκB proteins by upstream IκB kinases (IKKs), followed by its proteasomal degradation. These events allow for nuclear translocation of NF-κB, where it binds to specific DNA sequences in the promoters of target DNA, thereby facilitating transcription. ...
Simple Summary
The bone marrow (BM) microenvironment plays a crucial role in pathogenesis of multiple myeloma (MM), and delineation of the intracellular signaling pathways activated in the BM microenvironment in MM cells is essential to develop novel therapeutic strategies to improve patient outcome.
Abstract
The multiple myeloma (MM) bone marrow (BM) microenvironment consists of different types of accessory cells. Both soluble factors (i.e., cytokines) secreted from these cells and adhesion of MM cells to these cells play crucial roles in activation of intracellular signaling pathways mediating MM cell growth, survival, migration, and drug resistance. Importantly, there is crosstalk between the signaling pathways, increasing the complexity of signal transduction networks in MM cells in the BM microenvironment, highlighting the requirement for combination treatment strategies to blocking multiple signaling pathways.
... IGF-1 activates phosphatidylinositol-3-kinase (PI-3K)/Akt signaling cascade and mitogen-activated Downloaded from mjiri.iums.ac.ir at 1:32 IRST on Monday November 23rd 2020 protein kinase (MAPK). Activated Akt can suppress apoptosis through inhibiting the activation of the interleukin-1βconverting enzyme (28)-like proteases, glycogen synthase kinase 3 (GSK3) and the mammalian target of rapamycin (mTOR) (29)(30)(31). Over-activation of PI-3K/Akt triggers NF-κB signaling and accelerates the aging process while impairment of PI-3K/Akt signaling leads to activation of FoxO factors and extending the lifespan (32). ...
... In this regard, A six-year study of 32,826 nurses, indicated that highest levels of IGF-1 are associated with twoand-a-half times greater risk of colorectal cancer (33). Moreover, cell line studies have shown that interleukin-6 (IL-6) promote the proliferation of multiple myeloma (MM) cells and protect them against dexamethasone-induced apoptosis by means of IGF-1 (29). Besides that, IGF1 or IGF1R over activation lead to migration and motility of cancer cell through the extension of lamellipodia in neuroblastoma cell lines (34). ...
... IGF-1 activates phosphatidylinositol-3-kinase (PI-3K)/Akt signaling cascade and mitogen-activated protein kinase (MAPK). Activated Akt can suppress apoptosis through inhibiting the activation of the interleukin-1βconverting enzyme (28)-like proteases, glycogen synthase kinase 3 (GSK3) and the mammalian target of rapamycin (mTOR) (29)(30)(31). Over-activation of PI-3K/Akt triggers NF-κB signaling and accelerates the aging process while impairment of PI-3K/Akt signaling leads to activation of FoxO factors and extending the lifespan (32). ...
... In this regard, A six-year study of 32,826 nurses, indicated that highest levels of IGF-1 are associated with twoand-a-half times greater risk of colorectal cancer (33). Moreover, cell line studies have shown that interleukin-6 (IL-6) promote the proliferation of multiple myeloma (MM) cells and protect them against dexamethasone-induced apoptosis by means of IGF-1 (29). Besides that, IGF1 or IGF1R over activation lead to migration and motility of cancer cell through the extension of lamellipodia in neuroblastoma cell lines (34). ...
Background: Insulin-like growth factor1 (IGF1) is a polypeptide that structurally is similar to human pro-insulin, one of the factors that is altered in obesity and many related diseases, hence a large body of research devoted to evaluate it.
Methods: In this mini-review, we briefly explain the role of IGF1 in different conditions, including obesity, cardiovascular disease, and cancer through the results of review and original articles in both animal and human studies.
Results: The short-term metabolic effect of IGF-1 is insulin-like, and its long-term effect is growth factor-like. IGF1 has different roles in the initiation and progression of different diseases, because in some cases, the anti-apoptotic effect, can help cell survival while in others, it may lead to cancer or increment of adipocytes.
Conclusion: It is highly recommended to consider the different impacts of IGF1 in health and diseases prevention in future studies and interventions
... Anthracyclines, particularly doxorubicin, have been demonstrated to activate NF-κB and its pro-survival downstream targets, contributing to chemoresistance [87]. The methods by which doxorubicin stimulates NF-κB include activation via the IKK complex, the PI3K dependent pathway, as well as c-Abl kinase activity as reported in different studies [88]. Involvement of signaling pathways in chemoresistance is multifactorial. ...
Cancer therapy has advanced from tradition chemotherapy methods to targeted therapy, novel drug delivery mechanisms, combination therapies etc. Although several novel chemotherapy strategies have been introduced, chemoresistance still remains as one of the major barriers in cancer treatments. Chemoresistance can lead to relapse and hinder the development of improved clinical results for cancer patients, and this continues to be the major hurdle in cancer therapy. Anticancer drugs acquire chemoresistance through different mechanisms. Understanding these mechanisms is crucial to overcome and increase the efficiency of the cancer therapies that are employed. The potential molecular pathways behind chemoresistance include tumor heterogeneity, elevated drug efflux, multidrug resistance, interconnected signaling pathways, and other factors. To surpass this limitation, new clinical tactics are to be introduced. This review aims to compile the most recent information on the molecular pathways that regulate chemoresistance in cancers, which will aid in development of new therapeutic targets and strategies.
... The phosphatidylinositide 3-kinase (PI3K)/AKT-signaling pathway is one of the major survival signals in cancer cells. Phosphorylated, activated AKT inactivates proapoptotic proteins but induces the expression of antiapoptotic proteins, leading to the inhibition of apoptosis [51]. In accordance with the increased Noxa, decreased Mcl-1 and increased apoptosis of A549 cells, we have also found significantly decreased percentages of A549 cells expressing phosphorylated AKT, pAKT, after the treatments with 2 and 5 (Fig. 13A). ...
... It promotes bone degradation via TRAF6, p62, IkBα and CYLD signalling cascades [115]. Moreover, proteins like IGF1 produced in bone marrow are known to indirectly activate NF-kB in MM [116]. Pharmacological inhibition of NF-kB signalling by IKK2 inhibitors [117] and proteasomal inhibitors [118] overcomes cytoprotective effects of NFKB-mediated drug resistance, survival and growth. ...
Multiple myeloma (MM) is an aggressive cancer characterised by malignancy of the plasma cells and a rising global incidence. The gold standard for optimum response is aggressive chemotherapy followed by autologous stem cell transplantation (ASCT). However, majority of the patients are above 60 years and this presents the clinician with complications such as ineligibility for ASCT, frailty, drug-induced toxicity and differential/partial response to treatment. The latter is partly driven by heterogenous genotypes of the disease in different subpopulations. In this review, we discuss emerging single cell technologies and applications in MM, population pharmacogenetics of MM, resistance to chemotherapy, genetic determinants of drug-induced toxicity, molecular signal transduction, as well as the role(s) played by epigenetics and noncoding RNAs including microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) that influence the risk and severity of the disease. Taken together, our discussions further our understanding of genetic variability in ‘myelomagenesis’ and drug-induced toxicity, augment our understanding of the myeloma microenvironment at the molecular and cellular level and provide a basis for developing precision medicine strategies to combat this malignancy.
... It has been demonstrated that insulin-like growth factor-1 (IGF-1) decreases ischemic damage by reducing infarct volume and ameliorates behavioral neurological deficits (Liu et al., 2001). IGF-1 also promotes the survival of neurons and glial cells and regulates cell proliferation (Mitsiades et al., 2002). Additionally, IGF-1 inhibits apoptosis and protects neurons against oxidative stress and excitotoxicity (Kooijman et al., 2009;Liu et al., 2001). ...
Sertoli cells (SCs) may be a new candidate to decrease ischemic damage due to their ability to secrete factors that actively protect neurons and inhibit uncontrollable immune responses. Pre‑treatment with these cells was considered in the current study. SCs were injected into the right striatum in rats using the stereotaxic technique. Ten days after injection, middle cerebral artery occlusion surgery was performed. Following these procedures, neurological deficit scores, brain edema, blood‑brain barrier integrity, infarct volume, and the expression of apoptotic factors in the cortex, striatum, and piriform cortex‑amygdala were evaluated. Analysis showed that behavioral deficits, infarct volume, blood‑brain barrier permeability, and edema in the striatal area in the allograft group demonstrated a significant decrease compared to the control group. Additionally, analysis of the expression of caspase‑3 and Bcl‑2 proteins in the striatum indicated a remarkable reduction and increase, respectively, in the allograft group compared to the control group. According to the obtained results, one possible mechanism for the neuroprotection induced by SCs in an ischemic brain is the reduction of apoptotic factors.
... In human myeloma cells, such as U266, IM-9 and RPMI8226 cells, exposure to high Ca 2+ concentration augmented cell proliferation through CaSR on their surfaces, and further participate in a vicious cycle by expanding myeloma cell mass in destructive bone lesions [57]. Studies have reported that some basal or constitutive activity of MAPK, Interleukin-6 (IL-6), insulin-like growth factor-1 (IGF-1) or other signaling pathways have been found in these myeloma cells, which can promot cell proliferation [82,83]. This is how myeloma cells survive in bone marrow microenvironment, indicating that CaSR, at least in part, might mediate these survival signals to regulate the mitosis of myeloma cells. ...
Multiple myeloma (MM) is a common malignant tumor of plasma cells. Despite several treatment approaches in the past two decades, MM remains an aggressive and incurable disease in dire need of new treatment strategies. Approximately 70–80% of patients with MM have myeloma bone disease (MBD), often accompanied by pathological fractures and hypercalcemia, which seriously affect the prognosis of the patients. Calcium channels and transporters can mediate Ca ²⁺ balance inside and outside of the membrane, indicating that they may be closely related to the prognosis of MM. Therefore, this review focuses on the roles of some critical calcium channels and transporters in MM prognosis, which located in the plasma membrane, endoplasmic reticulum and mitochondria. The goal of this review is to facilitate the identification of new targets for the treatment and prognosis of MM.
... On the other hand, two studies by Mitsiades et al. reported contradictory results. Exposure of an MM cell line to the cytokine IGF-1 stimulated NFκB signaling, which was accompanied by upregulation of anti-apoptotic proteins including BFL-1 [35]. In addition, a specific NFκB inhibitor induced apoptosis in MM PC isolated from patients and cell lines. ...
Multiple myeloma (MM) is a hematological malignancy that is still considered incurable due to the development of therapy resistance and subsequent relapse of disease. MM plasma cells (PC) use NFκB signaling to stimulate cell growth and disease progression, and for protection against therapy-induced apoptosis. Amongst its diverse array of target genes, NFκB regulates the expression of pro-survival BCL-2 proteins BCL-XL, BFL-1, and BCL-2. A possible role for BFL-1 in MM is controversial, since BFL-1, encoded by BCL2A1, is downregulated when mature B cells differentiate into antibody-secreting PC. NFκB signaling can be activated by many factors in the bone marrow microenvironment and/or induced by genetic lesions in MM PC. We used the novel signal transduction pathway activity (STA) computational model to quantify the functional NFκB pathway output in primary MM PC from diverse patient subsets at multiple stages of disease. We found that NFκB pathway activity is not altered during disease development, is irrespective of patient prognosis, and does not predict therapy outcome. However, disease relapse after treatment resulted in increased NFκB pathway activity in surviving MM PC, which correlated with increased BCL2A1 expression in a subset of patients. This suggests that BFL-1 upregulation, in addition to BCL-XL and BCL-2, may render MM PC resistant to therapy-induced apoptosis, and that BFL-1 targeting could provide a new approach to reduce therapy resistance in a subset of relapsed/refractory MM patients.
... Paradoxically, ROS also promote tumor growth by acting as signals for proliferation. For example, the release of H 2 O 2 from mitochondria activates transcription factor NF-kB, which is activated by myeloma cells in order to increase proliferation and survival (100). ROS from the mitochondria may also activate the PI3K and MAPK pathways, which also promote tumorgenesis (101). ...
Multiple myeloma (MM) is an incurable cancer arising from malignant plasma cells that engraft in the bone marrow (BM). The physiology of these cancer cells within the BM microenvironment (TME) plays a critical role in MM development. These processes may be similar to what has been observed in the TME of other (non-hematological) solid tumors. It has been long reported that within the BM, vascular endothelial growth factor (VEGF), increased angiogenesis and microvessel density, and activation of hypoxia-induced transcription factors (HIF) are correlated with MM progression but despite a great deal of effort and some modest preclinical success the overall clinical efficacy of using anti-angiogenic and hypoxia-targeting strategies, has been limited. This review will explore the hypothesis that the TME of MM engrafted in the BM is distinctly different from non-hematological-derived solid tumors calling into question how effective these strategies may be against MM. We further identify other hypoxia-mediated effectors, such as hypoxia-mediated acidification of the TME, oxygen-dependent metabolic changes, and the generation of reactive oxygen species (ROS), that may prove to be more effective targets against MM.
... BAFF P. J.Hengeveld et al. have reported that BAFF can be used as a biomarker for myeloma burden and for the estimation of the progression of disease(83). Patients with myeloma and higher concentrations of BAFF show worse PFS (32), mostly as BAFF promote the survival of both B cell (immature, naive and activated B cells) and MM cell by active the NF-B pathway(84). High level of BAFF concentration can lead the proliferation of MM cell. ...
Despite new efficacy drugs and cell therapy have been used for multiple myeloma (MM) patients, some patients will relapse over time. We wonder the immune system play a vital role as well as MM cell during the development of disease. It is clear that the characteristic of myeloma cell is associated with the survival of MM patients. However, the link between the immune profiling and the prognosis of the disease is still not entirely clear. As more study focus on the role of immunity on multiple myeloma pathogenesis. There are plenty of study about the predictive role of immunity on the survival of multiple myeloma patients. Up to mow, the majority reviews published have focused on the immunotherapy and immune pathogenesis. It is indispensable to overlook the predictive role of immunity on multiple myeloma patients. Here, we give a review of vital previous works and recent progress related to the predictive role of immune profiling on multiple myeloma, such as absolute lymphocyte count, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, lymphocytes and cytokines.
... promoting GPR120-mediated gene activation that has an inhibitory action on phosphorylation of NFkB (101,123). Furthermore, NFkB can be activated by proinflammatory cytokines such as interleukins (IL) and tumor necrosis factor α (TNFα), which their production has been shown to decrease with dietary n-3 LCPs (101,(124)(125)(126). ...
Despite the significant progress the world has made over the past few decades, millions of children in low- and middle-income countries still suffer from poor growth and development. Growth faltering during the critical window period of the first 1,000 days after conception is associated with multiple adverse consequences limiting human potential and economic growth. Recognizing the magnitude of the problem and its severe consequences, the World Health Assembly in 2012 endorsed a Comprehensive Implementation Plan on Maternal, Infant, and Young Child Nutrition with six global targets, chief among which is the target to reduce the number of stunted children by 40% in 2025. Identifying priority areas of action in high nutrition-burden countries is the first step to accelerate current progress and achieve the global nutrition targets. For the purpose of facilitating evidence-based decision-making, the first part of this Ph.D. thesis explores nutrition-specific and -sensitive factors that can contribute to a reduction in chronic child undernutrition in low- and middle-income countries. We pooled data from 50 Demographic and Health Surveys conducted in 14 low- and middle-income countries to explain the trend in under-five stunting prevalence over the past two decades. A four-level mixed-effects linear probability model, accounting for clustering of data by sampling clusters, survey-rounds, and countries, was fitted to estimate the association between the change in a range of distal to proximal determinants at a country-level and stunting risk for an individual child while adjusting for time trend and child-level covariates. Furthermore, innovative approaches that maximize the impact of existing nutrition interventions are highly required to assist the progress in low- and middle-income countries. Complementary feeding interventions in low-income settings to date have been focused on the energy and micronutrient content of diets and yielded only small to moderate effects on growth and development. Increasing evidence support the hypothesis that environmental enteric dysfunction, chronic gut inflammation with morphological and functional derangements, and systemic inflammation occurring at a high prevalence in children living in poor settings, could be an important missing link that mediates and reduces the expected benefits from interventions. There is evidence that omega-3 long-chain polyunsaturated fatty acids (n-3 LCPs) may improve gut integrity, reduce inflammation and enhance maturation of the immune system, which could lead to amelioration of this condition and the associated growth impairment. Additionally, some studies in high-income populations showed that n-3 LCPs may have benefits for infant neurocognitive development. However, there is limited evidence from studies testing these potential benefits in infants and young children in a low-income setting. Therefore, in the second part of the PhD thesis, we hypothesized that an increased intake of n-3 LCPs would result in reduced morbidity and inflammation, and improved growth and development of children aged 6-24 months in a low-income setting. To test these proposed hypotheses, we conducted the OME3JIM project involving a 2 x 2 factorial randomized controlled trial of n-3 LCPs-rich fish-oil supplementation (500 mg/day n-3-LCPs) through lactation (MI), complementary feeding (CI), or a combination of both (MCI). We enrolled 360 pairs of lactating mothers and their infants 6-12 months old from three rural communities in Jimma district, southwest Ethiopia. The primary study outcomes were child linear growth, i.e., monthly changes in length-for-age z score (LAZ) over the 12 months intervention follow-up, and the evolution of developmental performance from baseline through 6 and 12 months of the intervention, using the Denver II and the Ages and Stages Questionnaire: Social Emotional tools. Secondary outcomes included LCP concentrations in maternal milk and child blood, anthropometry measurements of weight-for-length z score (WLZ), head-circumference and mid-upper arm circumference (MUAC), nutritional status (stunting, wasting and anemia), common childhood morbidities, and inflammation using C-reactive protein. Chapter 3 presents results of the study on the trend in child stunting. Stunting followed a declining trend in all the 14 countries studied at an average annual reduction rate of 1.04 percentage points (pp). Among the distal factors assessed, a decrease in the Gini coefficient, an improvement in women’s decision-making, and an increase in urbanization over time within a country were significantly associated with a lower risk of stunting. Improvements in households’ access to improved sanitation facilities and drinking water sources, and children’s access to basic vaccinations were the important intermediate service-related drivers of stunting risk identified, whereas an improvement in early initiation of breastfeeding and a decrease in the prevalence of low birthweight were the important proximal drivers. Our findings indicate that although there has been progress in reducing stunting, the rate of reduction in the studied countries was below the average 3.9 pp annual reduction rate required to meet the global target set for 2025. Furthermore, our findings reinforce the need for a combination of nutrition-specific and -sensitive interventions on top of economic development to tackle the problem of chronic childhood undernutrition. The identified drivers will help to guide global efforts to further accelerate stunting reduction and monitor progress against chronic child undernutrition. Results of the OME3JIM study are presented and discussed in Chapters 4-6. From the total of 360 mother-infant pairs enrolled, 87% completed all the 12 months study follow-ups and the mean (SD) duration of supplementation was 11.0 (2.9) months. Compliance rate for the child and the maternal interventions were ~80% and ~70%, with no difference between study arms. All statistical analyses were conducted following the intention-to-treat principle. In Chapter 4, we present the efficacy of fishoil supplementation of lactating mothers on human milk LCP concentrations using a random sub-sample of 154 study participants. Fish-oil supplementation during lactation increased maternal milk concentrations of docosahexaenoic acid (DHA) by 39.0% (P < 0.001) and eicosapentaenoic acid (EPA) by 36.2% (P < 0.001), whereas the ratio of arachidonic acid (AA)/(DHA + EPA) decreased by 53.5% (P < 0.001), compared to the control. However, the maternal milk DHA concentration still remained lower than international norms after the intervention. The results demonstrate that fish-oil supplementation during lactation improves n-3 LCPs status of the maternal milk. In these mothers with a very low baseline breastmilk DHA status, which further declines over the course of lactation, a higher dose of supplementation may be required to attain optimal breastmilk DHA levels. Chapter 5 presents the independent and combined effects of the fish-oil intervention through lactation and complementary food on child n-3 LCPs status, health and growth. Fish-oil supplementation significantly increased child blood n-3 LCPs concentrations (P < 0.01) and decreased the AA/(DHA + EPA) ratio (P < 0.001) in all the MI, CI and MCI intervention arms as compared to the control. Fish-oil intervention also resulted in a better ponderal growth of children, as indicated by the small, but statistically significant, positive effects on monthly WLZ changes in the CI (effect size: 0.022/month; 95% CI: 0.005, 0.039/month; P = 0.012) and MCI arms (effect size: 0.018/month; 95% CI: 0.001, 0.034/month; P = 0.041). We also noted a non-significant trend towards larger monthly MUAC increments in the CI and MCI arms compared to the control. No further effects were detected on the primary study outcome linear growth or on the other secondary outcomes of growth, nutritional status, morbidity, and inflammation. Chapter 6 presents the effects of the same intervention on child development performance. There was no difference between study arms on the evolution of overall and social-emotional developmental performance over time (intervention by time interaction: F = 1.09; P = 0.35, and F = 0.61; P = 0.61, respectively). Overall, the findings from the OME3JIM trial did not support our primary study hypotheses that dietary n-3 LCPs supplementation through lactation and/or complementary feeding improves linear growth and development of infants and young children from a rural setting in Ethiopia. n-3 LCP supplementation given directly to children or in combination with maternal supplementation was found to modestly increase relative weight gain. In conclusion, this PhD research provides evidence on a set of potentially important proximal to distal factors that can contribute to reduction in chronic childhood undernutrition in low- and middle-income countries. It also contributes to the limited literature on the effects of n-3 LCP supplementation in infants and young children in a rural sub-Saharan African setting. In Chapter 7 the implications of the study findings are discussed and recommendations for future research and policy are provided. It is underlined that economic development and nutrition-sensitive interventions, on top of nutrition-specific programs, could play an important role in further reduction of the high stunting burden in low- and middle-income countries. Future follow-up of the OME3JIM cohort is also recommended to determine whether there are long-term effects of the fish-oil intervention.
... Similar to IL-6, IGF-1 produced by MM-MSCs is another important mediator of PC growth, survival, migration, and drug resistance [98]. IGF-1 induced signaling pathways in myeloma cells (PI3K/AKT, MAPK, and NF-κB) that resulted in increased telomerase activity and upregulation of the antiapoptotic molecules, such as surviving cellular FADDlike IL-1β-converting enzyme (FLICE)-inhibitory protein (c-FLIP), X-linked inhibitor of apoptosis protein (XIAP), cellular inhibitor of apoptosis 2 (cIAP-2), and BCL-2-related protein A1 (BFL1) [99,100]. ...
Simple Summary
Multiple myeloma is a cancer of immunoglobulin-secreting cells that accumulate in the bone marrow. Mesenchymal stromal cells are important components of the bone marrow microenvironment interacting with myeloma cells and having a pivotal role in the progression of the disease. Here we first review studies that have highlighted structural and functional differences between mesenchymal stromal cells derived from healthy donors and myeloma patients, and propose a model for the transition from the normal to the myeloma-condition of these cells. Next, we underscore the contribution of mesenchymal stromal cells to the promotion of myeloma growth and survival, development of drug resistance, dissemination and homing, myeloma bone disease, and the establishment of a pro-inflammatory and immunosuppressive microenvironment. It appears as if as a result of myeloma-mesenchymal stromal cell cross-talk, mesenchymal stromal cells in myeloma patients have converted into active contributors to the pathophysiology of the disease.
Abstract
Multiple myeloma (MM) is a hematological malignancy of plasma cells that proliferate and accumulate within the bone marrow (BM). Work from many groups has made evident that the complex microenvironment of the BM plays a crucial role in myeloma progression and response to therapeutic agents. Within the cellular components of the BM, we will specifically focus on mesenchymal stromal cells (MSCs), which are known to interact with myeloma cells and the other components of the BM through cell to cell, soluble factors and, as more recently evidenced, through extracellular vesicles. Multiple structural and functional abnormalities have been found when characterizing MSCs derived from myeloma patients (MM-MSCs) and comparing them to those from healthy donors (HD-MSCs). Other studies have identified differences in genomic, mRNA, microRNA, histone modification, and DNA methylation profiles. We discuss these distinctive features shaping MM-MSCs and propose a model for the transition from HD-MSCs to MM-MSCs as a consequence of the interaction with myeloma cells. Finally, we review the contribution of MM-MSCs to several aspects of myeloma pathology, specifically to myeloma growth and survival, drug resistance, dissemination and homing, myeloma bone disease, and the induction of a pro-inflammatory and immunosuppressive microenvironment.
... The rationale for AKT-inhibition in MM is based on high levels of activation in MM cells when compared to cells from patients with MGUS or smoldering MM and inhibition leading to decreased viability in cell lines [71]. Interestingly mutations in the AKT-pathway are not commonly found in MM [72], suggesting an alternative mechanism of AKTpathway activation as a consequence of other deregulated pathways, such as the MAPK pathway, Il-6 signaling, or the NFκB network [73][74][75][76][77]. ...
Simple Summary
Multiple Myeloma is a cancer of plasma cells in the bone marrow. While effective treatments are available and many patients can now live years with the disease, most patients ultimately run out of treatment options. Pathway-directed therapy looks at genetic aberrations in the tumor cells and tries to blockade the tumor’s Achilles heel. Since myeloma cells show changes in several pathways, which can vary between different patients, agents targeting these pathways often only show activity in few patients. Pathway-directed therapy in myeloma is therefore often combined with personalized medicine, which aims to identify drugs that might interfere with the most important pathways in a particular patient. There are several pathways that can be targeted in myeloma, and, in combination with personalized medicine, some have shown promising results. However, there is still a challenge in identifying suitable patients and preventing resistance to single drugs, most likely caused by other pathways assuming the function of the blockaded one. Further research is therefore required to improve pathway-directed therapy.
Abstract
Multiple Myeloma (MM) is a malignant plasma cell disorder with an unmet medical need, in particular for relapsed and refractory patients. Molecules within deregulated signaling pathways, including the RAS/RAF/MEK/ERK, but also the PI3K/AKT-pathway belong to the most promising evolving therapeutic targets. Rationally derived compounds hold great therapeutic promise to target tumor-specific abnormalities rather than general MM-associated vulnerabilities. This paradigm is probably best depicted by targeting mutated BRAF: while well-tolerated, remarkable responses have been achieved in selected patients by inhibition of BRAFV600E alone or in combination with MEK. Targeting of AKT has also shown promising results in a subset of patients as monotherapy or to resensitize MM-cells to conventional treatment. Approaches to target transcription factors, convergence points of signaling cascades such as p53 or c-MYC, are emerging as yet another exciting strategy for pathway-directed therapy. Informed by our increasing knowledge on the impact of signaling pathways in MM pathophysiology, rationally derived Precision-Medicine trials are ongoing. Their results are likely to once more fundamentally change treatment strategies in MM.
... 48 Akt activation also drives nuclear factor (NF)-κB activation, which, in turn, controls the expression of the anti-apoptotic proteins FLICE-inhibitory protein (FLIP) and X-linked inhibitor of apoptosis protein (XIAP). 49 The main EMT-TFs responsible for migration, invasion or dedifferentiation also play a role in cell survival by modulating the expression of pro-and anti-apoptotic proteins. For example, Twist increases Bcl-2, leading to apoptotic resistance, 50 whereas SNAI1 interacts with poly(ADP-ribose) polymerase 1 (PARP1). ...
The continuing efforts to exploit the death receptor agonists, such as the tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), for cancer therapy, have largely been impaired by the anti-apoptotic and pro-survival signalling pathways leading to drug resistance. Cell migration, invasion, differentiation, immune evasion and anoikis resistance are plastic processes sharing features of the epithelial-to-mesenchymal transition (EMT) that have been shown to give cancer cells the ability to escape cell death upon cytotoxic treatments. EMT has recently been suggested to drive a heterogeneous cellular environment that appears favourable for tumour progression. Recent studies have highlighted a link between EMT and cell sensitivity to TRAIL, whereas others have highlighted their effects on the induction of EMT. This review aims to explore the molecular mechanisms by which death signals can elicit an increase in response heterogeneity in the metastasis context, and to evaluate the impact of these processes on cell responses to cancer therapeutics.
... In addition there is an interaction between NF-κB and FOXO, as a downstream mediator of the IIS pathway. The FOXO3A linked with longevity in humans was detected to prevent the activation of NF-κB [138,139] and FOXO3A knockout mice show overactivation of NF-κB especially in T-cell populations [140]. Among another aging-associated pathways that have been reported, NF-κB and mTOR signaling are connected. ...
Healthy aging and human longevity are intricate phenotypes affected by environmental factors such as
21 physical exercise, diet, health habits, and psychosocial situations as well as genetic factors. Diet and
22 caloric restriction have a crucial role in healthy aging. Curcumin, a polyphenolic compound isolated from
23 the Curcuma longa, has been shown to exert anti-aging characteristics. Recently, investigations on
24 curcumin with regard to aging and age-associated disease in model organisms has described that
25 curcumin and its metabolites, prolong the mean lifespan of some aging model organisms such as C.
26 elegans, D. melanogaster, yeast, and mouse. It has been proposed to have several biological activities,
27 such as antioxidative, anti-inflammatory, anticancer, chemopreventive, and anti-neurodegenerative
28 characteristics. In several studies on various model organisms it has been shown that the lifespan
29 extension via curcumin treatment was connected with enhanced superoxide dismutase (SOD) activity, and
30 also declined malondialdehyde (MDA) and lipofuscin levels. As well as the pivotal role of curcumin on
31 the modulating of major signaling pathways that influence longevity of organisms like IIS, mTOR, PKA,
32 and FOXO signaling pathways. This review defines the use of curcumin in traditional and modern
33 medicine, its biochemistry and biological functions, such as curcumin's anti-aging, anti-cancer, anti34 microbial, anti-inflammatory, and anti-oxidant characteristics. Also, the review further describes the role
35 of curcumin in a pharmacological context and new insights on its therapeutic capacity and restrictions.
36 Particularly, the review emphasizes in-depth on the efficiency of curcumin and its mechanism of action as
37 an anti-aging compound and also treating age-related disease.
... NF-κB-target genes also encode some of the VEGF isoforms. Furthermore, the NF-κB dependent antiapoptotic gene expression is caused by IGF-1, which is secreted by BMSCs [144,145]. The canonical NF-κB pathway is activated by TNF (a pro-inflammatory cytokine) in both myeloma cells as well as in BMSCs, and it is also secreted by the canonical pathway [2]. ...
Multiple myeloma (MM) is a hematologic disorder of B lymphocytes characterized by the accumulation of malignant plasma cells (PCs) in the bone marrow. The altered plasma cells overproduce abnormal monoclonal immunoglobulins and also stimulate osteoclasts. The host’s immune system and microenvironment are of paramount importance in the growth of PCs and, thus, in the pathogenesis of the disease. The interaction of MM cells with the bone marrow (BM) microenvironment through soluble factors and cell adhesion molecules causes pathogenesis of the disease through activation of multiple signaling pathways, including NF-κβ, PI3K/AKT and JAK/STAT. These activated pathways play a critical role in the inhibition of apoptosis, sustained proliferation, survival and migration of MM cells. Besides, these pathways also participate in developing resistance against the chemotherapeutic drugs in MM. The imbalance between inflammatory and anti-inflammatory cytokines in MM leads to an increased level of pro-inflammatory cytokines, which in turn play a significant role in dysregulation of signaling pathways and proliferation of MM cells; however, the association appears to be inadequate and needs more research. In this review, we are highlighting the recent findings on the roles of various cytokines and growth factors in the pathogenesis of MM and the potential therapeutic utility of aberrantly activated signaling pathways to manage the MM disease.
... IGF-1 has always been thought to serve as a proliferative and antiapoptotic factor in MM [12][13][14]. However, mounting recent evidence has shown that IGF-1 promotes migration and invasion in myeloma cells [15][16][17]. ...
... IGF-1 was reported to be important for cell survival by AKT signaling pathways, which further mediate NF-κB activation in MM cells. 25 Thus, there may be a negative feedback loop among NF-κB, PHLPP and Akt, which may lead to a mild effect on cell apoptosis. However, the precise mechanism needs more evidences to illustrate. ...
Introduction:
Treatment of bortezomib (BTZ) improves the clinical outcomes of patients with multiple myeloma (MM). However, primary resistance and acquired resistance to BTZ frequently develop in patients with MM. PH domain leucine-rich repeat protein phosphatase (PHLPP) plays an important role in chemoresistance in a number of cancers. However, the role of PHLPP on MM remains unclear. In this study, we investigated the role of PHLPP in BTZ-resistant MM cells.
Methods:
BrdU assays, immunoprecipitation, flow cytometry analyses, and immunofluorescence assays were performed.
Results:
PHLPP and lysosome-associated membrane protein 2 (LAMP2) levels were downregulated in BTZ-resistant MM cells compared with BTZ-sensitive MM cells, accompanied by inactivation of autophagy pathway evaluated by a reduction in Beclin1, Atg5 and LC3B and increase in p62. Gain- and loss-of-function experiments revealed that PHLPP partially re-sensitized MM cells to BTZ. In addition, PHLPP overexpression increased whereas PHLPP knockdown reduced LAMP2 expression, subsequently regulating the autophagy pathway in MM cells. Further findings demonstrated that LAMP2 knockdown reversed PHLPP-mediated cell apoptosis and autophagy activation in MM cells.
Conclusion:
This study demonstrated that PHLPP is a potential strategy for overcoming BTZ resistance in patients with MM.
... В пользу такого предположения свидетельствует тот факт, что в промоторной области гена MDR1 / ABCB1 имеется сайт связывания с транскрипционным фактором NF-κB, экспрессия которого регулируется IGF-1 [12][13][14]. Не исключено, что в микроокружении костного мозга IGF-1, продуцируемый фибробластами и остеобластами, воздействует на клетки ММ паракринным образом и активирует в них экспрессию транскрипционного фактора NF-κB, который, в свою очередь, активирует в клетках ММ экспрессию MDR1 / ABCB1. В литературе имеется несколько работ, посвященных изучению взаимосвязи между IGF-1 и возникновением лекарственной устойчивости к некоторым химиопрепаратам при ММ. ...
Introduction . Insulin-like growth factors (IGF) are one of the widely studied factors in oncology. For tumors with a high expression level of IGF typical postoperative relapse, they are invasive and give distant metastases. There are also data on the participation of IGF in the emergence of resistance to anticancer drugs. The mechanisms that determine the influence of insulin-like growth factors on the progression of a number of malignant neoplasms remain undisclosed and carrying out fundamental research in this direction is relevant. Objective: to study the role of IGF type 1 (IGF-1) in multiple myeloma (MM).Materials and methods . 26 samples of bone marrow aspirates received from 26 patients – 14 men and 12 women – were studied in the work. All patients were diagnosed with stage III ММ. The age of patients ranged from 52 to 72 years. From the obtained bone marrow aspirates, using centrifugation in the Ficoll gradient, a mononuclear fraction of bone marrow cells containing plasma cells was obtained. Then we carried out the procedure of extracting RNA and using polymerase chain reaction with reverse transcription, we studied the expression of mRNA of the genes of IGF-1 and MDR1/ABCB1.Results . The paper analyzes the overall survival (OS) of patients with MM depending on the expression of the gene IGF-1. It is shown that for patients with MM who have a high level of IGF-1 expression, a decrease in OS is characteristic and, conversely, with a weak expression of IGF-1 or in the absence of its expression, an increase in OS is observed. Studies of expression of IGF-1 gene and MDR1/ABCB1 gene responsible for the occurrence of multiple drug resistance showed that these genes are co-expressed in patients with MM. Conclusion . The obtained results indicate that the high level of IGF-1 gene expression may be a poor prognostic factor in ММ. IGF-1 may participate in regulation of the mechanisms of emergence of multiple drug resistance in patients with MM.
... The IGF-1 was reported to be even more important for survival of MM cells than IL-6 [97]. IGF-1 activates the mitogen-activated protein kinase (MAPK) and the phosphatidylinositol-3′-kinase (PI-3 K)/AKT signaling pathways in MM cells, which further mediate NF-κB activation and above-mentioned IL-6 signaling [98]. Another important growth factor regulated by NF-κB is BAFF, which in turn activates NF-κB, AKT and MAPK signaling pathways that further regulate expression of other survival factors, such as Bcl-2 [99]. ...
Multiple myeloma, which ranks as the second most common hematological malignancy, is known for its great genetic heterogeneity. One pathway, however, stands out in this diverse group. NF-κB pathway is one of the most important pathways in multiple myeloma not only for its role in pathogenesis, but also for its importance in various treatment strategies. Mutations in several major components of the NF-κB pathway and its regulators are present in at least 17% of primary multiple myeloma tumors and 42% of multiple myeloma cell lines. The NF-κB pathway regulates numerous genes, which influence development and pathogenesis of multiple myeloma. This significance of NF-κB for myeloma cells, however, is used against them, as current treatment strategies often use NF-κB as their primary or secondary target.
In order to discover new anticancer drugs, novel ruthenium(III) complexes [Ru(L)Cl(H2O)], where L is tetradentate Schiff base bis(acetylacetone)ethylendiimine (acacen, 1), bis(benzoylacetone)ethylendiimine (bzacen, 2), (acetylacetone)(benzoylaceton)ethylendiimine (acacbzacen, 3), bis(acetylacetone)propylendiimine (acacpn, 4), bis(benzoylacetone)propylendiimine (bzacpn, 5) or (acetylacetone)(benzoylaceton)propylendiimine (acacbzacpn, 6), were synthesized. The complexes 1–6 were characterized by elemental analysis, molar conductometry, and by various spectroscopic techniques, such as UV–Vis, IR, EPR, and ESI-MS. Based on in vitro DNA/BSA experiments, complexes 2 (bzacen) and 5 (bzacpn) with two aromatic rings showed the highest DNA/BSA-activity, suggesting that the presence of the aromatic ring on the tetradentate Schiff base ligand contributes to increased activity. Moreover, these two compounds showed the highest cytotoxic effects toward human, A549 and murine LLC1 lung cancer cells. These complexes altered the ratio of anti- and pro-apoptotic molecules and induced apoptosis of A549 cells. Further, complexes 2 and 5 reduced the percentage of Mcl1 and Bcl2 expressing LLC1 cells, induced their apoptotic death and exerted an antiproliferative effect against LLC1. Finally, complex 5 reduced the volume of mouse primary heterotopic Lewis lung cancer, while complex 2 reduced the incidence and mean number of metastases per lung. Additionally, molecular docking with DNA revealed that the reduced number of aromatic rings or their absence causes lower intercalative properties of the complexes in order: 2 > 5 > 6 > 3 > 4 > 1. It was observed that conventional hydrogen bonds and hydrophobic interactions contribute to the stabilization of the structures of complex-DNA. A molecular docking study with BSA revealed a predominance of 1–6 in binding affinity to the active site III, a third D-shaped hydrophobic pocket within subdomain IB.
Overview
Plasma cell disorders have in common a proliferation of monoclonal plasma cells associated with the production of a monoclonal protein. These disorders range from the common, indolent condition of monoclonal gammopathy of undetermined significance to malignancies, such as multiple myeloma, characterized by the presence of hypercalcemia, anemia, renal dysfunction, and/or lytic lesions. Progress in the understanding of the molecular underpinnings of myeloma has led to remarkable advances in its treatment. High‐dose melphalan with autologous stem‐cell transplant was historically a mainstay of treatment. Now, highly effective and well‐tolerated drug classes such as the proteasome inhibitors (e.g., bortezomib and carfilzomib) and immunomodulatory drugs (e.g., lenalidomide and pomalidomide) have rapidly transformed the treatment of myeloma and significantly improved the overall survival. The increasing use of extended treatment strategies such as maintenance therapy and the arrival of newer drug classes such as plasma cell‐specific monoclonal antibodies are setting the stage for improving outcomes further.
Multiple signaling pathways facilitate the survival and drug resistance of malignant B-cells by regulating their migration and adhesion to microenvironmental niches. NF-κB pathways are commonly dysregulated in mantle cell lymphoma (MCL), but the exact underlying mechanisms are not well understood. Here, using a co-culture model system, we show that the adhesion of MCL cells to stromal cells is associated with elevated levels of KDM6B histone demethylase mRNA in adherent cells. The inhibition of KDM6B activity, using either a selective inhibitor (GSK-J4) or siRNA-mediated knockdown, reduces MCL adhesion to stromal cells. We showed that KDM6B is required both for the removal of repressive chromatin marks (H3K27me3) at the promoter region of NF-κB encoding genes and for inducing the expression of NF-κB genes in adherent MCL cells. GSK-J4 reduced protein levels of the RELA NF-κB subunit and impaired its nuclear localization. We further demonstrated that some adhesion-induced target genes require both induced NF-κB and KDM6B activity for their induction (e.g., IL-10 cytokine gene), while others require induction of NF-κB but not KDM6B (e.g., CCR7 chemokine gene). In conclusion, KDM6B induces the NF-κB pathway at different levels in MCL, thereby facilitating MCL cell adhesion, survival, and drug resistance. KDM6B represents a novel potential therapeutic target for MCL.
Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of immunoglobulin-secreting clonal plasma cells at the bone marrow (BM). The interaction between MM cells and the BM microenvironment, and specifically BM mesenchymal stem cells (BM-MSCs), has a key role in the pathophysiology of this disease. Multiple data support the idea that BM-MSCs not only enhance the proliferation and survival of MM cells but are also involved in the resistance of MM cells to certain drugs, aiding the progression of this hematological tumor. The relation of MM cells with the resident BM-MSCs is a two-way interaction. MM modulate the behavior of BM-MSCs altering their expression profile, proliferation rate, osteogenic potential, and expression of senescence markers. In turn, modified BM-MSCs can produce a set of cytokines that would modulate the BM microenvironment to favor disease progression. The interaction between MM cells and BM-MSCs can be mediated by the secretion of a variety of soluble factors and extracellular vesicles carrying microRNAs, long non-coding RNAs or other molecules. However, the communication between these two types of cells could also involve a direct physical interaction through adhesion molecules or tunneling nanotubes. Thus, understanding the way this communication works and developing strategies to interfere in the process, would preclude the expansion of the MM cells and might offer alternative treatments for this incurable disease.
Purpose:
BRD9 is a defining component of the non-canonical SWI/SNF complex, which regulates gene expression by controlling chromatin dynamics. Although recent studies have found an oncogenic role for BRD9 in multiple cancer types including multiple myeloma (MM), its clinical significance and oncogenic mechanism have not yet been elucidated. Here, we sought to identify the clinical and biological impact of BRD9 in MM, which may contribute to the development of novel therapeutic strategies.
Experimental design:
We performed integrated analyses of BRD9 in vitro and in vivo using MM cell lines and primary MM cells in established preclinical models, which identified the molecular functions of BRD9 contributing to MM cell survival.
Results:
We found that high BRD9 expression was a poor prognostic factor in MM. Depleting BRD9 by genetic (shRNA) and pharmacological (dBRD9-A; proteolysis-targeting chimera; BRD9 degrader) approaches downregulated ribosome biogenesis genes, decreased the expression of the master regulator MYC, and disrupted the protein-synthesis maintenance machinery, thereby inhibiting MM cell growth in vitro and in vivo in preclinical models. Importantly, we identified that the expression of ribosome biogenesis genes was associated with the disease progression and prognosis of MM patients. Our results suggest that BRD9 promotes gene expression by predominantly occupying the promoter regions of ribosome biogenesis genes and cooperating with BRD4 to enhance the transcriptional function of MYC.
Conclusions:
Our study identifies and validates BRD9 as a novel therapeutic target in preclinical models of MM, which provides the framework for the clinical evaluation of BRD9 degraders to improve patient outcome.
Introduction:
Oftentimes, radiation therapy (RT) is not effective due to the development of radioresistance (RR). However, studies have shown that targeting epigenetic modifiers to enhance radiosensitivity represents a promising direction of clinical investigation.
Areas covered:
This review will first discuss the mechanisms by which epigenetic modifiers alter radiosensitivity. This is followed by a thorough discussion of how these modifiers promote or suppress RR through the dysregulation of MAPK-ERK and AKT-mTOR signaling. Finally, we discuss the clinical directions for targeting epigenetic modifiers as future therapeutic tools and current radiology techniques in the clinic.
Unlabelled:
We searched Pubmed and ScienceDirect databases from April 4th, 2022 to October 18th, 2022. We examined 226 papers based on title and abstract reviews related to radioresistance, epigenetics, MAPK, and PI3K/AKT/mTOR signaling. 194 papers were selected for this review. Keywords used for this search include, "radioresistance", "radiosensitivity", "radiation", "radiotherapy", "particle radiation", "photon radiation", "epigenetic modifiers", "MAPK", "AKT", "mTOR", "cancer", and "PI3K". Further, we examined 41 papers related to clinical trials on the aforementioned topics. The outcomes of interest were safety, overall survival (OS), dose-limiting toxicities (DLT), progression-free survival (PFS), and maximum tolerated dose (MTD).
Expert opinion:
Current studies focusing on epigenetic mechanisms of RR strongly support the use of targeting epigenetic modifiers as adjuvants to standard cancer therapies. To further the success of such treatments and their clinical benefit for future patients, both preclinical and clinical studies are needed to broaden the scope of known radioresistant mechanisms.
Propofol is an important and widely used anaesthetic drug in the clinic. Many works have shown that propofol has important biological functions except as an anaesthetic. In the current study, we mainly explored the effect of propofol on the biological activity of IGF-1, which is an important growth factor involved in regulating the growth and development of the stomach. Here, we explored the effect of propofol on the biological activity of IGF-1 in a GES-1-cell model. We found that propofol affected the biological activity of IGF-1. It not only reduces IGF-1/IGF-1R signalling but also changes IGF-1R cell characteristics. We further explored the mechanism by which propofol affected IGF-1 activity. Through a series of experiments, we found that propofol affected the stability of membrane-localised IGF-1R. It also affects the recycling of the IGF-1R receptor Propofol can affect the degradation of IGF-1R by changing the endocytosis of IGF-1R. In short, the current study found that propofol affected the biological activity of IGF-1, which laid the foundation for related research.
Introduction: Thyroid carcinoma (THCA) is the most common endocrine tumor worldwide. Insulin-like growth factor 1 (IGF1) is a polypeptide hormone with a high degree of structural similarity to human proinsulin. Materials and Methods: We used online data to investigate the expression pattern of IGF1 in THCA. We also identified gene signatures and pathways downstream of IGF1 to elucidate potential mechanisms. Further analysis identified critical hub genes and pathways using the STRING database and the DAVID online tool. We analyzed gene expression and the prognostic quality of hub genes using the GEPIA online tool and the Human Protein Atlas website. Results: IGF1 expression was found to be downregulated in THCA tissues, both those in the TCGA database and in tissues recovered during surgery at our hospital. All samples from the THCA-TCGA dataset were divided into IGF1 high- and low-expression groups. We identified 1014 differentially expressed genes (DEGs) between the two groups. Functional enrichment analysis showed that DEGs were mainly concentrated in immune response, cell adhesion, and cancer-related pathways. We then identified the top hub genes and performed a prognostic analysis to identify the most critical genes. The expression levels of FN1, MMP9, and CD40LG correlated with disease prognosis. Finally, we confirmed the expression levels of these genes using the HPA website. Conclusion: Our results identified potential key genes and pathways downstream of IGF1 in THCA, providing insight into the mechanisms underlying the development of THCA.
Multiple myeloma is increasingly being recognised as more than one disease, characterised by marked cytogenetic, molecular, and proliferative heterogeneity. The prognosis is widely varied, ranging from low to very high-risk, based on cytogenetic and molecular studies. Although novel agents, such as proteasome inhibitors and immunomodulators, have been developed, which have improved treatment responses and disease prognosis, multiple myeloma remains an incurable disease. Based on highly sensitive detection tools, such as gene expression profiling and next generation sequence analysis, and the understanding of the pathogenesis of multiple myeloma, many potential agents, including monoclonal antibodies, drug-conjugated antibodies, drugs targeted to molecular abnormalities, microRNA inhibitors or mimics, and immune therapies, such as chimeric antigen receptors T cells and anti-PD1 agents, can be considered personalised therapies. In this paper, multiple myeloma pathogenesis and potential molecular and immunotherapies are reviewed.
In Multiple Myeloma (MM) the finely tuned homeostasis of the bone marrow (BM) microenvironment is disrupted. Evasion of programmed cell death (apoptosis) represents a hallmark of cancer. Besides genetic aberrations, the supportive and protective MM BM milieu, which is constituted by cytokines and growth factors, intercellular and cell: extracellular matrix (ECM) interactions and exosomes, in particular, plays a key role in the abundance of pro-survival members of the Bcl-2 family (i.e., Mcl-1, Bcl-2, and Bcl-xL) in tumor cells. Moreover, microenvironmental cues have also an impact on stability- regulating post-translational modifications of anti-apoptotic proteins including de/phosphorylation, polyubiquitination; on their intracellular binding affinities, and localization.
Advances of our molecular knowledge on the escape of cancer cells from apoptosis have informed the development of a new class of small molecules that mimic the action of BH3-only proteins. Indeed, approaches to directly target anti-apoptotic Bcl-2 family members are among today’s most promising therapeutic strategies and BH3-mimetics (i.e., venetoclax) are currently revolutionizing not only the treatment of CLL and AML, but also hold great therapeutic promise in MM. Furthermore, approaches that activate apoptotic pathways indirectly via modification of the tumor microenvironment have already entered clinical practice.
The present review article will summarize our up-to-date knowledge on molecular mechanisms by which the MM BM microenvironment, cytokines, and growth factors in particular, mediates tumor cell evasion from apoptosis. Moreover, it will discuss some of the most promising science- derived therapeutic strategies to overcome Bcl-2- mediated tumor cell survival in order to further improve MM patient outcome.
Overview
Plasma cell disorders have in common a proliferation of monoclonal plasma cells associated with the production of a monoclonal protein. These disorders range from the common, indolent condition of monoclonal gammopathy of undetermined significance to malignancies such as multiple myeloma characterized by the presence of hypercalcemia, anemia, renal dysfunction, and/or lytic bone lesions. Progress in the understanding of the molecular underpinnings of myeloma has led to remarkable advances in its treatment. High‐dose melphalan and autologous stem cell transplant have been a mainstay of treatment. Now, highly effective and well‐tolerated drug classes such as the proteasome inhibitors (e.g., bortezomib, carfilzomib), immunomodulatory drugs (e.g., lenalidomide, pomalidomide), and anti‐CD38 monoclonal antibodies (e.g., daratumumab, isatuximab) have rapidly transformed treatment and significantly improved overall survival. Newer therapies targeting B‐cell maturation antigen (BCMA) have the potential to further improve outcomes.
Resistance to therapeutic treatment and metastatic progression jointly determine a fatal outcome of cancer. Cancer metastasis and therapeutic resistance are traditionally studied as separate fields using non-overlapping strategies. However, emerging evidence, including from in vivo imaging and in vitro organotypic culture, now suggests that both programmes cooperate and reinforce each other in the invasion niche and persist upon metastatic evasion. As a consequence, cancer cell subpopulations exhibiting metastatic invasion undergo multistep reprogramming that — beyond migration signalling — supports repair programmes, anti-apoptosis processes, metabolic adaptation, stemness and survival. Shared metastasis and therapy resistance signalling are mediated by multiple mechanisms, such as engagement of integrins and other context receptors, cell–cell communication, stress responses and metabolic reprogramming, which cooperate with effects elicited by autocrine and paracrine chemokine and growth factor cues present in the activated tumour microenvironment. These signals empower metastatic cells to cope with therapeutic assault and survive. Identifying nodes shared in metastasis and therapy resistance signalling networks should offer new opportunities to improve anticancer therapy beyond current strategies, to eliminate both nodular lesions and cells in metastatic transit.
Aim: The present study intended to compare the antioxidant, anti-lipid peroxidation, and anti-inflammatory potentials of Nigella Sativa (NS) and onion extract on 5-FU-induced liver damage in rats. Material and methods: 48 rats were divided into control, control group of the onion extract, control group of the NS extract, 5-FU-treated, concomitant NS-treated, and concomitant onion extract-treated. Liver sections were processed for histological analysis (light and electron microscopic examination). Liver enzymes (ALT, AST, and ALP), inflammatory markers (TNF-α and IL-1), antioxidant markers (SOD, GSH, and GSH/GSSG ratio), 4-HNE, NF-κB, and Nrf2 were evaluated. Results: The 5-FU-treated group exhibited inflammation, congested hepatic sinusoid, and steatosis. Improvement with few pathological residues was seen in the concomitant extract-treated groups. The 5-FU-treated group showed higher liver enzymes. The enzymes decreased in the concomitantly treated groups. 5-FU induced liver damage through oxidative stress, inflammation, and lipid peroxidation. Concomitantly using NS and onion extracts resulted in a reduction in oxidative stress, lipid peroxidation, and inflammation. Conclusion: NS and onion extracts attenuated 5-FU-induced liver damage via antioxidative, anti-lipid peroxidative, and anti-inflammatory mechanisms. NS’s role was exceptional when compared with onion extract.
In recent years, the immune system has increasingly been recognized as a critical component to understanding cancer progression and cellular microenvironments. This has led to massive growth in the field of cancer immunotherapy, in which the patient's natural defense mechanisms are harnessed and enhanced to fight cancer. In this field, T-cell engineering has been the most widely studied and developed approach, with checkpoint inhibitors like anti-PD-1 antibody recently showing impressive clinical success for solid tumors and ex vivo engineering of chimeric antigen receptor T-cells making a significant impact on liquid tumors. There has been continued basic and translational research interest in developing engineering technologies for delivery of therapeutic agents to induce T-cell reprogramming both ex vivo and in vivo for cancer immunotherapy. In this chapter, delivery technologies that provide surface stimulation to T-cells (outside-in) as well as technologies that deliver intracellular mediators to T-cells (inside-out) for reprogramming to enhance anti-cancer activity are discussed.
Immunoglobulin light-chain (AL) amyloidosis is a rare life-threatening disease caused by light chains that are toxic to vital organs such as the heart, kidneys, liver and peripheral nervous system, and that misfold and assemble as amyloid fibrils and deposit both in affected organs and systemically in the vasculature and other tissues. Patients afflicted by this disease have B-cell disorders, almost always related to clonal plasma cells in the bone marrow, the burden of which can range from small clones involving 5% or less of marrow cells to frank multiple myeloma. The goal of therapy is to eliminate the clonal plasma cells producing these toxic light chains to halt and possibly reverse symptomatic organ damage. While autologous stem cell transplantation can be a very effective treatment modality in AL, it has a limited role due to the frailty of this particular population. Conservative treatment in the form of chemotherapy has become the backbone of therapy. Bortezomib combined with alkylators has proven quite successful in inducing hematologic responses. However, despite these advances, tolerability and resistance continue to be an ongoing issue. Novel anti-plasma cell therapies such as ixazomib, carfilzomib, lenalidomide and pomalidomide are actively being combined and evaluated in clinical trials for efficacy and toxicity in this challenging patient population. Other approaches, such as monoclonal antibodies targeting surface proteins and amyloid deposits, are being tested and combined with novel agents. In this review, we will provide an overview of the clinical trials that have led to current treatment algorithms and will also discuss monoclonal antibodies currently under investigation and in various stages of clinical development.
The systematic shortening of the non-covalent element of a C8-linked pyrrolobenzodiazepine (PBD) conjugate (13) led to the synthesis of a 19-member library of C8-PBD monomers. The critical elements of 13, which were required to render the molecule cytotoxic, were elucidated by an annexin V assay. The effects of shortening the non-covalent element of the molecule on transcription factor inhibitory capacity were also explored through an ELISA-based measurement of nuclear NF-κB upon exposure of JJN3 cells to the synthesised molecules. While shortening the non-covalently interactive element of 13 had a lesser than expected effect upon compound cytotoxicity due to reduced DNA interaction, the transcription factor inhibitory capacity of the molecule was notably altered. This study suggests that a relatively short non-covalent side chain at the C8-position of PBD is sufficient to confer cytotoxicity. The shortened PBD monomers provide a new ADC payload scaffold due to their potent cytotoxicity and drug-like properties.
Multiple myeloma (MM) is an incurable plasma cell malignancy. Aberrant activation of the Hedgehog (Hh) and NF-κB signaling pathways is observed in MM and plays a pivotal role in the development of MM by promoting myeloma cell growth, survival, and drug resistance. In this study, we found that the Sonic Hh (SHh) ligand in the bone marrow microenvironment is responsible for the enhancement of NF-κB activity in MM cell lines NCI-H929 and U266. Notably, we discovered that Hh signaling regulates NF-κB through its classical pathway (SHh/PTCH1/SMO/GLI1) in MM cells. Meanwhile, non-classical pathway by SMO recruitment of TRAF6 to ubiquitination is also involved in it. Moreover, the SMO inhibitor cyclopamine enhances the cytotoxic effects of bortezomib in MM cell lines. Our study reveals the cross-talk between Hh members and the NF-κB pathway in the myeloma cells and provides a theoretical basis for combined utilization of Hh members and proteasome inhibition in MM.
BACKGROUND: Multiple myeloma (MM) is the second most common hematological malignancy with an incidence of 4.3 per 100,000 in the general population, and a median survival of 3-5 years (1). It is a malignancy of the plasma cells, usually immature plasmablasts that exhibit chromosomal abnormalities. These early chromosomal abnormalities, such as translocations, result in overexpression of several important oncogenes (2-4). Eventually, malignant clones carrying these mutations progress and undergo further genetic insults leading to advanced disease. Environmental factors play an equally important role in the progression of the disease (Fig. 1). The malignant plasma cells home to the bone marrow (BM) where subsequent interactions promote MM cell growth, survival, and migration, as well as the development of drug resistance. The BM microenvironment (BMM) consists of extracellular matrix proteins such as laminin, collagen, fibronectin and osteopontin (5). The complex landscape of cells in the BMM includes hematopoietic stem cells, BM stromal cells (BMSC), BM endothelial cells, fibroblasts, osteoclasts and osteoblasts. Within the BMM, multiple signaling pathways and proteins are dysregulated in MM compared to normal plasma cells (6), including PI3K/Akt, MAPK, JAK/STAT, IKK/IkB/NF-kB, and HSP90, which therefore represent novel targets in MM (Fig. 2).
Among cancers of different origin, multiple myeloma (MM) has carved out a position of prominence, as an effective model system to test and validate leading therapeutic compounds. Indeed, several drugs now in clinical practice were first successfully tested in MM, and then their use extended to other hematological and epithelial tumors.
Tumor necrosis factor superfamily member TRAIL/Apo-2L has recently been shown to induce apoptosis in transformed and cancer cells. Some prostate cancer cells express constitutively active Akt/protein kinase B due to a complete loss of lipid phosphatase PTEN gene, a negative regulator of phosphatidylinositol 3-kinase pathway. Constitutively active Akt promotes cellular survival and resistance to chemotherapy and radiation. We have recently noticed that some human prostate cancer cells are resistant to TRAIL. We therefore examined the intracellular mechanisms of cellular resistance to TRAIL. The cell lines expressing the highest level of constitutively active Akt were more resistant to undergo apoptosis by TRAIL than those expressing the lowest level. Down-regulation of constitutively active Akt by phosphatidylinositol 3-kinase inhibitors, wortmannin and LY294002, reversed cellular resistance to TRAIL. Treatment of resistant cells with cycloheximide (a protein synthesis inhibitor) rendered cells sensitive to TRAIL. Transfecting dominant negative Akt decreased Akt activity and increased TRAIL-induced apoptosis in cells with high Akt activity. Conversely, transfecting constitutively active Akt into cells with low Akt activity increased Akt activity and attenuated TRAIL-induced apoptosis. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage, as caspase-8 activity was not affected. Enforced expression of anti-apoptotic protein Bcl-2 or Bcl-X(L) inhibited TRAIL-induced mitochondrial dysfunction and apoptosis. We therefore identify Akt as a constitutively active kinase that promotes survival of prostate cancer cells and demonstrate that modulation of Akt activity, by pharmacological or genetic approaches, alters the cellular responsiveness to TRAIL. Thus, TRAIL in combination with agents that down-regulate Akt activity can be used to treat prostate cancer.
To control agonist-induced nuclear translocation of transcription factor kappa B (NF-kappa B) in intact cells, cell-permeable synthetic peptides were devised. Their import into intact cells was dependent on a hydrophobic region selected from the signal peptide sequences and was verified by their inaccessibility to extracellular proteases and by confocal laser scanning microscopy. When a cell-permeable peptide carried a functional cargo representing the nuclear localization sequence of NF-kappa B p50, it inhibited in a concentration-dependent manner nuclear translocation of NF-kappa B in cultured endothelial and monocytic cells stimulated with lipopolysaccharide or tumor necrosis factor-alpha. Synthetic peptide analogues with deleted hydrophobic cell membrane-permeable motif or with a mutated nuclear localization sequence were inactive. Cell membrane-permeable peptides were not cytotoxic within the concentration range used in these experiments. These results suggest that cell-permeable synthetic peptides carrying a functional cargo can be applied to control signal transduction-dependent subcellular traffic of transcription factors mediating the cellular responses to different agonists. Moreover, this approach can be used to study other intracellular processes involving proteins with functionally distinct domains.
AP-1-associated factor 1 (AF-1), is a novel protein complex that dramatically enhances the assembly of JunD-containing dimers
onto AP-1 consensus sites. We describe the partial purification of AF-1 from nuclear extracts of the T-cell line MLA 144 by
ionic, hydrophobic and gel filtration chromatography. AF-1 is a DNA-binding protein composed of low molecular mass polypeptides
of 7-17 kDa that exists in solution as a 34-kDa complex. JunD interactions with DNA are accelerated in the presence of AF-1
through the formation of a true tri-molecular complex with JunD dimers and DNA that assembles much more rapidly on DNA than
JunD alone. DNA binding analysis of AF-1 interaction with JunD·AP-1 and DNA shows that AF-1 increases the DNA binding affinity
of JunD for AP-1 sites over 100-fold. DNA cleavage footprint analysis of isolated AF-1·JunD DNA complexes shows that the ternary
complex makes nearly twice as many contacts with DNA than JunD dimers alone. AF-1 interacts readily, but differentially with
Jun homodimers and Jun·Fos heterodimers. These findings distinguish AF-1 as a significant protein-specific modulator of AP-1·JunD
in T-cells.
The role of insulin-like growth factor 1 (IGF-1) for the treatment of neurodegenerative disorders, such as Alzheimer's disease, has recently gained attention. The present study demonstrates that IGF-1 promotes the survival of rat primary cerebellar neurons and of immortalized hypothalamic rat GT1-7 cells after challenge with oxidative stress induced by hydrogen peroxide (H2O2). Neuroprotective concentrations of IGF-1 specifically induce the transcriptional activity and the DNA binding activity of nuclear factor kappaB (NF-kappaB), a transcription factor that has been suggested to play a neuroprotective role. This induction is associated with increased nuclear translocation of the p65 subunit of NF-kappaB and with degradation of the NF-kappaB inhibitory protein IkappaBalpha. IGF-1-mediated protection of GT1-7 cells against oxidative challenges was mimicked by overexpression of the NF-kappaB subunit c-Rel. Partial inhibition of NF-kappaB baseline activity by overexpression of a dominant-negative IkappaBalpha mutant enhanced the toxicity of H2O2 in GT1-7 cells. The pathway by which IGF-1 promotes neuronal survival and activation of NF-kappaB involves the phosphoinositol (PI) 3-kinase, because both effects of IGF-1 are blocked by LY294002 and wortmannin, two specific PI 3-kinase inhibitors. Taken together, our results provide evidence for a novel molecular link between IGF-1-mediated neuroprotection and induction of NF-kappaB that is dependent on the PI 3-kinase pathway.
The molecular mechanisms by which multiple myeloma (MM) cells evade glucocorticoid-induced apoptosis have not been delineated. Using a human IgAkappa MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-kappaB DNA binding and kappaB-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-kappaB complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of NF-kappaB DNA binding precedes a notable increase in annexin V binding, thereby indicating that diminished NF-kappaB activity is an early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1 cells prevents Dex-mediated repression of NF-kappaB activity and apoptosis. Sustained NF-kappaB DNA binding is also observed in two previously characterized Dex-resistant MM cell lines (RPMI8226 and ARH-77) that express moderate levels of endogenous bcl-2 and IkappaBalpha proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not prevent the augmentation of IkappaBalpha protein by Dex. We also noted a possible association between Dex-mediated downregulation of NF-kappaB in freshly obtained primary myeloma cells and the patients' responsiveness to glucocorticoid-based chemotherapy. Collectively, our data suggest that the protective effects of bcl-2 in MM cells act upstream in the NF-kappaB activation-signaling pathway and the potential use of NF-kappaB as a biomarker in progressive MM.
Nuclear factor kappaB (NF-kappaB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-kappaB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induced time-dependent NF-kappaB nuclear translocation. The nuclear NF-kappaB protein after QA treatment was comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein were markedly increased at the time of QA-induced NF-kappaB nuclear translocation. Immunohistochemical analysis showed that c-Myc and p53 induction occurred in the excitotoxin-sensitive medium-sized striatal neurons. NF-kappaB nuclear translocation was blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-kappaB SN50, but not by the NF-kappaB SN50 control peptide. NF-kappaB SN50 significantly inhibited the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-kappaB SN50, but not the control peptide, also substantially reduced the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-kappaB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating an unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis.
NF-kappa B is involved in the transcriptional control of various genes that act as extrinsic and intrinsic survival factors for T cells. Our findings show that suppression of NF-kappa B activity with cell-permeable SN50 peptide, which masks the nuclear localization sequence of NF-kappa B1 dimers and prevents their nuclear localization, induces apoptosis in resting normal human PBL. Inhibition of NF-kappa B resulted in the externalization of phosphatidylserine, induction of DNA breaks, and morphological changes consistent with apoptosis. DNA fragmentation was efficiently blocked by the caspase inhibitor Z-VAD-fmk and partially blocked by Ac-DEVD-fmk, suggesting that SN50-mediated apoptosis is caspase-dependent. Interestingly, apoptosis induced by NF-kappa B suppression, in contrast to that induced by TPEN (N,N,N',N'-tetrakis [2-pyridylmethyl]ethylenediamine) or soluble Fas ligand (CD95), was observed in the absence of active death effector proteases caspase-1-like (IL-1 converting enzyme), caspase-3-like (CPP32/Yama/apopain), and caspase-6-like and without cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and DNA fragmentation factor-45. These findings suggest either low level of activation is required or that different caspases are involved. Preactivation of T cells resulting in NF-kappa B nuclear translocation protected cells from SN50-induced apoptosis. Our findings demonstrate an essential role of NF-kappa B in survival of naive PBL.
TNF and Fas ligand induce apoptosis in tumor cells; however, their severe toxicity toward normal tissues hampers their application to cancer therapy. Apo2 ligand (Apo2L, or TRAIL) is a related molecule that triggers tumor cell apoptosis. Apo2L mRNA is expressed in many tissues, suggesting that the ligand may be nontoxic to normal cells. To investigate Apo2L's therapeutic potential, we generated in bacteria a potently active soluble version of the native human protein. Several normal cell types were resistant in vitro to apoptosis induction by Apo2L. Repeated intravenous injections of Apo2L in nonhuman primates did not cause detectable toxicity to tissues and organs examined. Apo2L exerted cytostatic or cytotoxic effects in vitro on 32 of 39 cell lines from colon, lung, breast, kidney, brain, and skin cancer. Treatment of athymic mice with Apo2L shortly after tumor xenograft injection markedly reduced tumor incidence. Apo2L treatment of mice bearing solid tumors induced tumor cell apoptosis, suppressed tumor progression, and improved survival. Apo2L cooperated synergistically with the chemotherapeutic drugs 5-fluorouracil or CPT-11, causing substantial tumor regression or complete tumor ablation. Thus, Apo2L may have potent anticancer activity without significant toxicity toward normal tissues.
Resistance of cancer cells against apoptosis induced by death factors contributes to the limited efficiency of immune- and drug-induced destruction of tumors. We report here that insulin and insulin-like growth factor-I (IGF-I) fully protect HT29-D4 colon carcinoma cells from IFN-gamma/tumor necrosis factor-alpha (TNF) induced apoptosis. Survival signaling initiated by IGF-I was not dependent on the canonical survival pathway involving phosphatidylinositol 3'-kinase. In addition, neither pp70(S6K) nor protein kinase C conveyed IGF-I antiapoptotic function. Inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) with the MAPK/ERK kinase inhibitor PD098059 and MAPK/p38 with the specific inhibitor SB203580 partially reversed, in a nonadditive manner, the IGF-I survival effect. Inhibition of nuclear factor kappaB (NF-kappaB) activity by preventing degradation of the inhibitor of NF-kappaB (IkappaB-alpha) with BAY 11-7082 also blocked in part the IGF-I antiapoptotic effect. However, the complete reversal of the IGF-I effect was obtained only when NF-kappaB and either MAPK/ERK or MAPK/p38 were inhibited together. Because these pathways are also those used by TNF to signal inflammation and survival, these data point to a cross talk between IGF-I- and TNF-induced signaling. We further report that TNF-induced IL-8 production was indeed strongly enhanced upon IGF-I addition, and this effect was totally abrogated by both MAPK and NF-kappaB inhibitors. The IGF-I antiapoptotic function was stimulus-dependent because Fas- and IFN/Fas-induced apoptosis was not efficiently inhibited by IGF-I. This was correlated with the weak ability of Fas ligation to enhance IL-8 production in the presence or absence of IGF-I. These findings indicate that the antiapoptotic function of IGF-I in HT29-D4 cells is based on the enhancement of the survival pathways initiated by TNF, but not Fas, and mediated by MAPK/p38, MAPK/ERK, and NF-kappaB, which act in concert to suppress the proapoptotic signals. In agreement with this model, we show that it was possible to render HT29-D4 cells resistant to Fas-induced apoptosis provided that IGF-I and TNF receptors were activated simultaneously.
Our previous studies have shown that activation of a related adhesion focal tyrosine kinase (RAFTK) (also known as Pyk2) is required for dexamethasone (Dex)-induced apoptosis in multiple myeloma (MM) cells and that human interleukin-6 (IL-6), a known growth and survival factor for MM cells, blocks both RAFTK activation and apoptosis induced by Dex. However, the mechanism whereby IL-6 inhibits Dex-induced apoptosis is undefined. In this study, we demonstrate that protein-tyrosine phosphatase SHP2 mediates this protective effect. We show that IL-6 triggers selective activation of SHP2 and its association with RAFTK in Dex-treated MM cells. SHP2 interacts with RAFTK through a region other than its Src homology 2 domains. We demonstrate that RAFTK is a direct substrate of SHP2 both in vitro and in vivo, and that Tyr(906) in the C-terminal domain of RAFTK mediates its interaction with SHP2. Moreover, overexpression of dominant negative SHP2 blocked the protective effect of IL-6 against Dex-induced apoptosis. These findings demonstrate that SHP2 mediates the anti-apoptotic effect of IL-6 and suggest SHP2 as a novel therapeutic target in MM.
A new human myeloma cell line, OPM-6, was established from the peripheral blood of a patient with advanced IgG-kappa plasma cell leukemia. Cytogenetic and phenotypic analysis confirmed that the cells were derived from the patient's leukemic cells. Insulin-like growth factor-1 (IGF-1) acts as an autocrine growth factor in these cells. In addition, OPM-6 cells were particularly sensitive to dexamethasone (DEX), when endogenous IGF-1 was blocked. Under these conditions, >95% of the DEX-treated cells died within 36 h. Therefore, OPM-6 represents a potentially powerful tool for the analysis of the molecular mechanisms of DEX-induced apoptosis, because it is possible to easily analyze the direct effects of DEX using this system. Using this culture system of OPM-6, we demonstrated that the treatment with DEX plus a monoclonal antibody to the human IGF-1 receptor (alphaIGF-1R) leads to the down-regulation of the gene expression of Bcl-xL, an antiapoptotic gene, and the activation of CPP32 during this apoptotic process. IFN-alpha as well as IL-6 prevented DEX plus alphaIGF-1R-induced apoptosis, and this prevention was blocked by the mitogen-activated protein kinase kinase inhibitor, PD098059, or the phosphatidylinositol 3-kinase inhibitor, wortmannin. Therefore, both IL-6 and IFN-alpha blocked DEX plus alphaIGF-1R-induced apoptosis through activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase pathways.
Astrocytes represent the most abundant cell type of the adult nervous system. Under normal conditions, astrocytes participate in neuronal feeding and detoxification. However, following brain injury, local increases in inflammatory cytokines trigger a reactive phenotype in astrocytes during which these cells produce their own inflammatory cytokines and neurotoxic free radicals. Indeed, progression of this inflammatory reaction is responsible for most neurological damage associated with brain trauma. Insulin-like growth factor-I (IGF-I) protects neurons against a variety of brain pathologies associated with glial overproduction of proinflammatory cytokines. Here, we demonstrate that in astrocyte cultures IGF-I regulates NFkappaB, a transcription factor known to play a key role in the inflammatory reaction. IGF-I induces a site-specific dephosphorylation of IkappaBalpha (phospho-Ser(32)) in astrocytes. Moreover, IGF-I-mediated dephosphorylation of IkappaBalpha protects this molecule from tumor necrosis factor alpha (TNFalpha)-stimulated degradation; therefore, IGF-I also inhibits the nuclear translocation of NFkappaB (p65) induced by TNFalpha exposure. Finally, we show that dephosphorylation of IkappaBalpha by IGF-I pathways requires activation of calcineurin. Activation of this phosphatase is independent of phosphatidylinositol 3-kinase and mitogen-activated protein kinase. Thus, these data suggest that the therapeutic benefits associated with IGF-I treatment of brain injury are derived from both its positive effects on neuronal survival and inhibition of the glial inflammatory reaction.
More than 80% of human ovarian cancers express LHRH and its receptor as part of a negative autocrine mechanism of growth control. This study was conducted to investigate whether LHRH affects apoptosis in ovarian cancer. EFO-21 and EFO-27 ovarian cancer cells were treated with LHRH agonist Triptorelin or with cytotoxic agent Doxorubicin in the absence or presence of Triptorelin. Apoptotic cells were quantified by flow cytometry. Expression of nuclear factor kappa B (NFkappaB) was assessed by RT-PCR and immunoblotting. For determination of Triptorelin-induced NFkappaB activation, cells were transfected with a NFkappaB-secreted alkaline phosphatase reporter gene plasmid (pNFkappaB-SEAP) and cultured for 96 h with or without Triptorelin. The causal relation between Triptorelin-induced NFkappaB activation and Triptorelin-induced protection against apoptosis was investigated using SN50, an inhibitor for nuclear translocation of activated NFkappaB. Apoptosis induction by Triptorelin was never observed. Treatment with Doxorubicin (1 nmol/L) for 72 h increased the percentage of apoptotic cells in EFO-21 and EFO-27 ovarian cancer cell lines to 31% or 34%, respectively. In cultures treated simultaneously with Triptorelin (100 nmol/L), the percentage of apoptotic cells was reduced significantly, to 17% or 18%, respectively (P < 0.001). RT-PCR and immunoblotting experiments showed that NFkappaB subunits p50 and p65 were expressed by ovarian cancer cell lines EFO-21 and EFO-27. When EFO-21 or EFO-27 cells were transfected with pNFkappaB-SEAP and subsequently treated with Triptorelin (100 nmol/L), NFkappaB-induced SEAP expression increased 5.3-fold or 4.7-fold, respectively (P < 0.001). Triptorelin-induced reduction of Doxorubicin-induced apoptosis was blocked by SN50-mediated inhibition of NFkappaB translocation into the nucleus. We conclude that LHRH induces activation of NFkappaB and thus reduces Doxorubicin-induced apoptosis in human ovarian cancer cells. This possibility to protect ovarian cancer cells from programmed cell death is an important feature in LHRH signaling in ovarian tumors, apart from the inhibitory interference with the mitogenic pathway.
The caspase-8 homologue FLICE-inhibitory protein (FLIP) functions as a caspase-8 dominant negative, blocking apoptosis induced by the oligomerization of the adapter protein FADD/MORT-1. FLIP expression correlates with resistance to apoptosis induced by various members of the tumor necrosis factor family such as TRAIL. Furthermore, forced expression of FLIP renders cells resistant to Fas-mediated apoptosis. Although FLIP expression is regulated primarily by MEK1 activity in activated T cells, the oncogenic signaling pathways that regulate FLIP expression in tumor cells are largely unknown. In this report, we examined the roles of the MAP kinase and phosphatidylinositol (PI) 3-kinase signaling pathways in the regulation of FLIP expression in tumor cells. We observed that the MEK1 inhibitor PD98059 reduced FLIP levels in only 2 of 11 tumor cell lines tested. In contrast, disruption of the PI 3-kinase pathway with the specific inhibitor LY294002 reduced Akt (protein kinase B) phosphorylation and the levels of FLIP protein and mRNA in all cell lines evaluated. The introduction of a dominant negative Akt adenoviral construct also consistently reduced FLIP expression as well as the phosphorylation of the Akt target glycogen synthase kinase-3. In addition, infection of the same cell lines with a constitutively active Akt adenovirus increased FLIP expression and the phosphorylation of GSK-3. These data add FLIP to the growing list of apoptosis inhibitors in which expression or function is regulated by the PI 3-kinase-Akt pathway.
We find that the prostate cancer cell lines ALVA-31, PC-3, and DU 145 are highly sensitive to apoptosis induced by TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand), while the cell lines TSU-Pr1 and JCA-1 are moderately sensitive, and the LNCaP cell line is resistant. LNCaP cells lack active lipid phosphatase PTEN, a negative regulator of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and demonstrate a high constitutive Akt activity. Inhibition of PI 3-kinase using wortmannin and LY-294002 suppressed constitutive Akt activity and sensitized LNCaP cells to TRAIL. Treatment of LNCaP cells with TRAIL alone induced cleavage of the caspase 8 and XIAP proteins. However, processing of BID, mitochondrial release of cytochrome c, activation of caspases 7 and 9, and apoptosis did not occur unless TRAIL was combined with either wortmannin, LY-294002, or cycloheximide. Blocking cytochrome c release by Bcl-2 overexpression rendered LNCaP cells resistant to TRAIL plus wortmannin treatment but did not affect caspase 8 or BID processing. This indicates that in these cells mitochondria are required for the propagation rather than the initiation of the apoptotic cascade. Infection of LNCaP cells with an adenovirus expressing a constitutively active Akt reversed the ability of wortmannin to potentiate TRAIL-induced BID cleavage. Thus, the PI 3-kinase-dependent blockage of TRAIL-induced apoptosis in LNCaP cells appears to be mediated by Akt through the inhibition of BID cleavage.
The caspase 8 homologue FLICE-inhibitory protein (cFLIP) is a potent negative regulator of death receptor-induced apoptosis.
We found that cFLIP can be upregulated in some cell lines under critical involvement of the NF-κB pathway, but NF-κB activation
was clearly not sufficient for cFLIP induction in all cell lines. Treatment of SV80 cells with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG-132) or geldanamycin, a drug interfering with tumor necrosis factor (TNF)-induced NF-κB activation,
inhibited TNF-induced upregulation of cFLIP. Overexpression of a nondegradable IκBα mutant (IκBα-SR) or lack of IκB kinase
γ expression completely prevented phorbol myristate acetate-induced upregulation of cFLIP mRNA in Jurkat cells. These data
point to an important role for NF-κB in the regulation of the cFLIP gene. SV80 cells normally show resistance to TNF-related
apoptosis-inducing ligand (TRAIL) and TNF, as apoptosis can be induced only in the presence of low concentrations of cycloheximide
(CHX). However, overexpression of IκBα-SR rendered SV80 cells sensitive to TRAIL-induced apoptosis in the absence of CHX,
and cFLIP expression was able to reverse the proapoptotic effect of NF-κB inhibition. Western blot analysis further revealed
that cFLIP, but not TRAF1, A20, and cIAP2, expression levels rapidly decrease upon CHX treatment. In conclusion, these data
suggest a key role for cFLIP in the antiapoptotic response of NF-κB activation.
Fas-mediated apoptosis proceeds though mitochondria-dependent or -independent pathways and is deficient in drug-resistant cells. Neuroblastoma, a common pediatric malignancy, often develops drug-resistance and has a silenced caspase 8 (FLICE) gene, which has been associated with Fas- and drug-resistance. We report that besides caspase 8, which was absent in approximately one-third of 26 neuroblastoma cases in this study, other proteins such as bcl-2 and FLICE-inhibitory protein (FLIP), are equally important in conferring Fas-resistance to neuroblastoma cells. Both bcl-2 and FLIP were frequently expressed in neuroblastoma tissues. Our in vitro studies showed that FLIP was recruited to the death-inducing signaling complex and interfered with the recruitment of caspase 8 in neuroblastoma cells. bcl-2 inhibited the activation of the mitochondria; but it also lowered the free cytoplasmic levels of caspase 8 by binding and sequestering it, thus acting through a novel antiapoptotic mechanism upstream of the mitochondria. In vitro down-regulation of bcl-2 with antisense oligonucleotides allowed the release of cytochrome c from mitochondria and the activation of caspases 8 and 3 upon Fas activation as well as sensitized neuroblastoma cells to Fas-mediated apoptosis. Down-regulation of FLIP had only a modest apoptotic effect because of the coexistent mitochondrial block. However, combined treatment with bcl-2 and FLIP antisense oligonucleotides had a statistically significant synergistic effect reversing Fas-resistance in neuroblastoma cells in vitro. These data indicate that Fas-mediated apoptosis in neuroblastoma cells is mitochondria-dependent and inhibited both at the mitochondrial level and at the level of caspase 8 activation. Thus, gene-targeting therapies for bcl-2 and FLIP may reverse Fas-resistance and prove useful in the treatment of drug-resistant neuroblastomas.
The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cell lines, whereas normal cells appear to be protected from its cytotoxic effects. Therefore TRAIL holds promise as a potential therapeutic agent against cancer. To elucidate some of the critical factors that contribute to TRAIL resistance, we performed a genetic screen in the human colon carcinoma cell line SW480 by infecting this TRAIL-sensitive cell line with a human placental cDNA retroviral library and isolating TRAIL-resistant clones. Characterization of the resulting clones for inhibitors of TRAIL-induced death (ITIDs) led to the isolation of c-FLIP(S), Bax inhibitor 1, and Bcl-XL as candidate suppressors of TRAIL signaling. We have demonstrated that c-FLIP(S) and Bcl-XL are sufficient when overexpressed to convey resistance to TRAIL treatment in previously sensitive cell lines. Furthermore both c-FLIP(S) and Bcl-XL protected against overexpression of the TRAIL receptors DR4 and KILLER/DR5. When c-FLIP(S) and Bcl-XL were overexpressed together in SW480 and HCT 116, an additive inhibitory effect was observed after TRAIL treatment suggesting that these two molecules function in the same pathway in the cell lines tested. Furthermore, we have demonstrated for the first time that a proapoptotic member of the Bcl-2 family, Bax, is required for TRAIL-mediated apoptosis in HCT 116 cells. Surprisingly, we have found that the serine/threonine protein kinase Akt, which is an upstream regulator of both c-FLIP(S) and Bcl-XL, is not sufficient when overexpressed to protect against TRAIL in the cell lines tested. These results suggest a key role for c-FLIP(S), Bcl-XL, and Bax in determining tumor cell sensitivity to TRAIL.
TRAIL/Apo-2L is a member of the tumor necrosis factor superfamily and has recently been shown to induce apoptosis in cancer cells, but not in normal cells. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without side effects. Akt promotes cell survival and block apoptosis. Some prostate cancer cells express high levels of Akt due to lack of active lipid phosphatase PTEN, a negative regulator of PI-3 kinase pathway, which may be responsible for drug resistance. The objective of this paper is to investigate the intracellular molecules that regulate TRAIL resistance. We have examined caspase-8 activity, BID cleavage, Akt activity, mitochondrial membrane potential (DeltaPsi(m)) and apoptosis in prostate cancer (LNCap, PC-3, PC-3M and DU145) cells treated with or without TRAIL. PC-3, PC-3M and DU145 cells are sensitive to TRAIL, whereas LNCap cells are resistant. LNCap cells express the highest level of constitutively active Akt, which is directly correlated with TRAIL resistance. TRAIL activates caspase-8 in all the cell lines. Downregulation of constitutively active Akt by PI-3 kinase inhibitors (wortmannin and LY-294002), dominant negative Akt or PTEN, renders LNCap cells sensitive to TRAIL. Inhibition of TRAIL sensitivity occurs at the level of BID cleavage. Inhibition of protein synthesis by cycloheximide also causes LNCap cells sensitive to TRAIL. Overexpression of Bcl-2 or Bcl-X(L) inhibits TRAIL-induced DeltaPsi(m) and apoptosis. Overexpression of constitutively active Akt in PC-3M cells (express very low levels of constitutively active Akt) restores TRAIL resistance. These data suggest that elevated Akt activity protects LNCap cells from TRAIL-induced apoptosis, and the PI-3 kinase/Akt pathway may inhibit apoptotic signals by inhibiting processing of BID. Thus, constitutively active Akt is an important regulator of TRAIL sensitivity in prostate cancer.
Our previous studies have characterized Dexamethasone (Dex)-induced apoptotic signaling pathways in multiple myeloma (MM) cells; however, related transcriptional events are not fully defined. In the present study, gene expression profiles of Dex-treated MM cells were determined using oligonucleotide arrays. Dex triggers early transient induction of many genes involved in cell defense/repair-machinery. This is followed by induction of genes known to mediate cell death and repression of growth/survival-related genes. The molecular and genetic alterations associated with Dex resistance in MM cells are also unknown. We compared the gene expression profiles of Dex-sensitive and Dex-resistant MM cells and identified a number of genes which may confer Dex-resistance. Finally, gene profiling of freshly isolated MM patient cells validates our in vitro MM cell line data, confirming an in vivo relevance of these studies. Collectively, these findings provide insights into the basic mechanisms of Dex activity against MM, as well as mechanisms of Dex-resistance in MM cells. These studies may therefore allow improved therapeutic uses of Dex, based upon targeting genes that regulate MM cell growth and survival.
The molecular mechanisms by which multiple myeloma (MM) cells evade glucocorticoid-induced apoptosis have not been delineated. Using a human IgAκ MM cell line (ARP-1), we found that dexamethasone (Dex)-induced apoptosis is associated with decreased NF-κB DNA binding and κB-dependent transcription. Both nuclear p50:p50 and p50:p65 NF-κB complexes are detected in ARP-1 cells by supershift electrophoretic mobility shift assay (EMSA). Dex-mediated inhibition of NF-κB DNA binding precedes a notable increase in annexin V binding, thereby indicating that diminished NF-κB activity is an early event in Dex-induced apoptosis. Overexpression of bcl-2 in ARP-1 cells prevents Dex-mediated repression of NF-κB activity and apoptosis. Sustained NF-κB DNA binding is also observed in two previously characterized Dex-resistant MM cell lines (RPMI8226 and ARH-77) that express moderate levels of endogenous bcl-2 and IκB proteins. In addition, enforced bcl-2 expression in ARP-1 cells did not prevent the augmentation of IκB protein by Dex. We also noted a possible association between Dex-mediated downregulation of NF-κB in freshly obtained primary myeloma cells and the patients’ responsiveness to glucocorticoid-based chemotherapy. Collectively, our data suggest that the protective effects of bcl-2 in MM cells act upstream in the NF-κB activation–signaling pathway and the potential use of NF-κB as a biomarker in progressive MM.
Cytokines in the tumor necrosis factor (TNF) family regulate development and function of the immune system. We have isolated a new member of this family, designated Apo-2 ligand (Apo-2L), via an expressed sequence tag. Apo-2L is a 281-amino acid protein, related most closely to Fas/Apo-1 ligand. Transfected Apo-2L is expressed at the cell surface with its C terminus exposed, indicating a type II transmembrane protein topology. Like Fas/Apo-1 ligand and TNF, the C-terminal extracellular region of Apo-2L (amino acids 114-281) exhibits a homotrimeric subunit structure. Soluble Apo-2L induces extensive apoptosis in lymphoid as well as non-lymphoid tumor cell lines. The effect of Apo-2L is not inhibited by soluble Fas/Apo-1 and TNF receptors; moreover, expression of human Fas/Apo-1 in mouse fibroblasts, which confers sensitivity to induction of apoptosis by agonistic anti-Fas/Apo-1 antibody, does not confer sensitivity to Apo-2L. Hence, Apo-2L acts via a receptor which is distinct from Fas/Apo-1 and TNF receptors. These results suggest that, along with other family members such as Fas/Apo-1 ligand and TNF, Apo-2L may serve as an extracellular signal that triggers programmed cell death.
Members of the NF-kappa B/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor alpha (TNF). Although NF-kappa B appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-kappa B is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-kappa B and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of Ikappa B that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-kappa B induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-kappa B via an Ikappa B targeting mechanism. Functional coupling of NF-kappa B and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.
Although X-linked inhibitor of apoptosis protein (Xiap) is an im- portant intracellular suppressor of apoptosis in a variety of cell types and is present in ovary, its physiological role in follicular development remains unclear. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor ne- crosis factor-a (TNFa) in rat granulosa cells. Granulosa cells from equine CG-primed immature rats were plated in RPMI 1640 medium containing 10% FCS and subsequently cultured in serum-free RPMI in the absence or presence of TNFa (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 mM), and/or adenoviral Xiap sense or an- tisense complementary DNA. TNFa alone failed to induce granulosa cell death, but in the presence of cycloheximide, it markedly increased the number of apoptotic granulosa cells (as assessed by in situ ter- minal deoxynucleotidyl transferase-mediated deox-UTP- biotin end labeling and DNA fragmentation analysis). Western anal- ysis indicated that TNFa alone increased the Xiap protein level, a response significantly reduced by adenoviral Xiap antisense expres- sion. Down-regulation of Xiap expression by antisense complemen- tary DNA induced granulosa cell apoptosis, which was potentiated by the cytokine. Inhibition of nuclear factor-kB activation by N-acetyl- cysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFa. The latter phenomenon was readily at- tenuated by adenoviral Xiap sense expression. In conclusion, these findings suggest that Xiap is an important intracellular modulator of the TNFa death signaling pathway in granulosa cells. Its expression is regulated by the TNFa via a nuclear factor-kB-mediated mecha- nism. (Endocrinology 142: 557-563, 2001)
Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homolgy (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC50 values in the 1−10 μM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.
Vanadium-induced TNF production is believed to play an important role in respiratory disease associated with air pollution and occupational exposure. While vanadium is able to induce TNF in macrophages or airway epithelial cells, the underlying mechanism is not well defined. In the present study, mechanisms of vanadate-induced TNF production were analyzed in the murine Raw264.7 cells. Vanadate induces a significant amount of TNF at both the protein and mRNA levels, and the induction is vanadate dose-dependent. The mechanism analysis was focused on transcriptional regulation of TNF gene by vanadate. Transient transfection studies show that the TNF gene promoter was activated by vanadate and this activation was associated with an increase in DNA binding activity of the nuclear factor-B (NF-B). Mutation of the NF-B binding site in the gene promoter led to a loss of the promoter responsiveness to vanadate, indicating requirement of NF-B. This is supported by evidence that inhibition of NF-B activation by SN50, a specific NF-B inhibitor, resulted in a decrease in the TNF production. A role of reactive oxygen species (ROS) was explored in vanadate activity. The result shows that vanadate-induced TNF production is elevated by NADPH, which enhances vanadate-mediated generation of ROS, but is inhibited by an antioxidant, N-acetyl-L-cysteine (NAC). Modification of TNF production is associated with an enhancement or a repression of NF-B activity by NADPH or NAC, respectively. Taken together, these results indicate that: (a) activation of the TNF gene promoter contributes to the vanadate-induced TNF production; (b) NF-B is required for the vanadate-induced promoter activity of TNF gene; (c) free radical reactions are involved in the vanadate-induced TNF production and NF-B activation.
Recent evidence indicates that the transcription factor NF-κB is a major effector of inducible antiapoptotic mechanisms. For example, it was shown that NF-κB activation suppresses the activation of caspase 8, the apical caspase in tumor necrosis factor (TNF) receptor family signaling cascades, through the transcriptional regulation of certain TRAF and IAP proteins. However, it was unknown whether NF-κB controls other key regulatory mechanisms in apoptosis. Here we show that NF-κB activation suppresses mitochondrial release of cytochrome
c
through the activation of the Bcl-2 family member A1/Bfl-1. The restoration of A1 in NF-κB null cells diminished TNF-induced apoptosis by reducing the release of proapoptotic cytochrome
c
from mitochondria. In addition, A1 potently inhibited etoposide-induced apoptosis by inhibiting the release of cytochrome
c
and by blocking caspase 3 activation. Our findings demonstrate that A1 is an important antiapoptotic gene controlled by NF-κB and establish that the prosurvival function of NF-κB can be manifested at multiple levels.
A novel tumor necrosis factor (TNF) family member has been cloned and characterized. This protein, designated TNF-related apoptosis-inducing ligand (TRAIL), consists of 281 and 291 aa in the human and murine forms, respectively, which share 65% aa identity. TRAIL is a type II membrane protein, whose C-terminal extracellular domain shows clear homology to other TNF family members. TRAIL transcripts are detected in a variety of human tissues, most predominantly in spleen, lung, and prostate. The TRAIL gene is located on chromosome 3 at position 3q26, which is not close to any other known TNF ligand family members. Both full-length cell surface expressed TRAIL and picomolar concentrations of soluble TRAIL rapidly induce apoptosis in a wide variety of transformed cell lines of diverse origin.
Human multiple myeloma (MM) represents a highly aneuploid tumor as shown by cytogenetic studies. This may partly explain the heterogeneity with regard to growth factor requirements demonstrated among MM cells. We have previously reported the expression of insulin-like growth factor I (IGF-I) and IGF-I receptor (IGF-IR) mRNA in some MM cell lines. In this study we investigated the role of IGF-I as a growth and/or survival factor in three MM cell lines: LP-1, EJM, and Karpas 707. We report that all cell lines expressed IGF-I and IGF-IR mRNA and protein. LP-1 and Karpas 707, but not EJM, were stimulated to proliferation in a dose-dependent manner by exogenous IGF-I. An IGF-IR blocking antibody inhibited both the IGF-I-induced and spontaneous growth of LP-1, and Karpas 707, while the EJM cell line was unaffected by the addition of the antibody. In conclusion, our results show that IGF-I can act as a growth factor in human MM, and they suggest that an autocrine IGF-I loop may contribute to the growth and survival in some MM cell lines.
Multiple myeloma cell lines express functional receptors for insulin-like growth factors (IGFs) and several cell types that make up the bone marrow microenvironment produce these cytokines. This suggests that IGFs may play a role in survival and/or expansion of the malignant clone within the marrow in patients with multiple myeloma. We tested the effects of these growth factors on myeloma cells challenged with dexamethasone. Dye exclusion and MTT assays demonstrated that both IGF-I and IGF-II protected the 8226 and dox-40 myeloma cell lines and three primary myeloma cultures from dexamethasone-induced cytotoxicity in a dose-dependent fashion. Morphologic studies of target cells and their nuclei as well as DNA electrophoresis confirmed the IGFs afforded protection against dexamethasone-induced apoptosis. Insulin also protected but was less impressive and required much higher concentrations. IGFs also protected against cycloheximide-induced apoptosis but were ineffective against serum starvation, topoisomerase II inhibitors, or anti-fas antibodies. IGF-induced protection against dexamethasone was not associated with any alteration in quantitative or qualitative expression of BCL-2, BAX or BCL-X proteins. These data indicate that insulin-like growth factors may play a role in maintenance of the malignant clone in patients with myeloma by protecting tumour cells from apoptotic death.
Insulin-like growth factors (IGF-I, IGF-II) have long been recognized as important mitogens in many types of malignancies. Because the role of IGFs in growth control of myeloma cells has not been extensively examined, we have used a panel of IL-6-responsive myeloma cell lines to address this issue. Initial studies demonstrated that IGF-I and IGF-II significantly enhanced DNA synthesis by each of the four cell lines, even when assayed in the absence of IL-6. The specificity of the IGF response was confirmed using an IGF-I receptor Ab, and additional studies demonstrated that IGF responsiveness did not result from induction of autocrine IL-6 expression. When IL-6 responsiveness was assayed, three of four cell lines synthesized DNA in response to IL-6 alone; however, the magnitude of responsiveness was greatly enhanced by addition of IGFs. Similar results were obtained when proliferation and cell cycle progression were analyzed. By contrast, the KP-6 cell line was responsive to IL-6 only when IGF was present. Finally, we analyzed the effects of IGF-I on normal B lymphocytes. IGF, however, did not stimulate B cell DNA synthesis, suggesting that IGF responsiveness may represent a key difference between normal and malignant B cells. In summary, these studies suggest that IGFs may play an important role in multiple myeloma by virtue of their ability to directly stimulate tumor cell growth as well as modulate the magnitude of IL-6-driven growth.
Excitotoxin-induced destruction of striatal neurons, proposed as a model of Huntington's disease, involves a process having the biochemical stigmata of apoptosis. Recent studies suggested that transcription factor nuclear factor (NF)-kappa B may be involved in excitotoxicity. To further analyze the contribution of NF kappa B to excitotoxic neuronal death in vivo, changes in binding activities of NF kappa B and other transcription factors as well as the consequences of inhibiting NF kappa B nuclear translocation were measured after the infusion of quinolinic acid (120 nmol) into rat striatum. Internucleosomal DNA fragmentation and terminal transferase-mediated dUTP digoxigenin nick end labeling-positive nuclei appeared 12 hr later and intensified over the next 12 hr. NF kappa B binding activity increased several-fold from 2 to 12 hr, then gradually declined during the next 12 hr. Other transcription factor changes included AP-1, whose binding peaked about 6 hr after quinolinic acid administration, and E2F-1, which was only modestly and transiently elevated. In contrast, quinolinic acid lead to a reduction in OCT-1, beginning after 12 hr, and briefly in SP-1 binding. The NF kappa B, AP-1, and OCT-1 changes were attenuated both by the N-methyl-D-aspartate receptor antagonist MK-801 and the protein synthesis inhibitor cycloheximide. Moreover, quinolinic acid-induced internucleosomal DNA fragmentation and striatal cell death were significantly reduced by the intrastriatal administration of NF kappa B SN50, a cell-permeable recombinant peptide that blocks NF kappa B nuclear translocation. These results illustrate the complex temporal pattern of transcription factor change attending the apoptotic destruction produced in rat striatum by quinolinic acid. They further suggest that NF kappa B activation contributes to the excitotoxin-induced death of striatal neurons.
Tumor necrosis factor α (TNF-α) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription
factor nuclear factor kappa B (NF-κB), which suppresses apoptosis by an unknown mechanism. The activation of NF-κB was found
to block the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins
c-IAP1 and c-IAP2 were identified as gene targets of NF-κB transcriptional activity. In cells in which NF-κB was inactive,
all of these proteins were required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 were sufficient to
suppress etoposide-induced apoptosis. Thus, NF-κB activates a group of gene products that function cooperatively at the earliest
checkpoint to suppress TNF-α–mediated apoptosis and that function more distally to suppress genotoxic agent–mediated apoptosis.
The serine/threonine kinase Akt (also known as protein kinase B, PKB) is activated by numerous growth-factor and immune receptors through lipid products of phosphatidylinositol (PI) 3-kinase. Akt can couple to pathways that regulate glucose metabolism or cell survival [1]. Akt can also regulate several transcription factors, including E2F, CREB, and the Forkhead family member Daf-16 [2] [3] [4]. Here, we show that Akt can regulate signaling pathways that lead to induction of the NF-kappaB family of transcription factors in the Jurkat T-cell line. This induction occurs, at least in part, at the level of degradation of the NF-kappaB inhibitor IkappaB, and is specific for NF-kappaB, as other inducible transcription factors are not affected by Akt overexpression. Furthermore, the effect requires the kinase activity and pleckstrin homology (PH) domain of Akt. Also, Akt does not act alone to induce cytokine promoters and NF-kappaB reporters, because signals from other pathways are required to observe the effect. These studies uncover a previously unappreciated connection between Akt and NF-kappaB induction that could have implications for the control of T-cell growth and survival.
Vanadium-induced TNFalpha production is believed to play an important role in respiratory disease associated with air pollution and occupational exposure. While vanadium is able to induce TNFalpha in macrophages or airway epithelial cells, the underlying mechanism is not well defined. In the present study, mechanisms of vanadate-induced TNFalpha production were analyzed in the murine Raw264.7 cells. Vanadate induces a significant amount of TNFalpha at both the protein and mRNA levels, and the induction is vanadate dose-dependent. The mechanism analysis was focused on transcriptional regulation of TNFalpha gene by vanadate. Transient transfection studies show that the TNFalpha gene promoter was activated by vanadate and this activation was associated with an increase in DNA binding activity of the nuclear factor-kappaB (NF-kappaB). Mutation of the NF-kappaB binding site in the gene promoter led to a loss of the promoter responsiveness to vanadate, indicating requirement of NF-kappaB. This is supported by evidence that inhibition of NF-kappaB activation by SN50, a specific NF-kappaB inhibitor, resulted in a decrease in the TNFalpha production. A role of reactive oxygen species (ROS) was explored in vanadate activity. The result shows that vanadate-induced TNFalpha production is elevated by NADPH, which enhances vanadate-mediated generation of ROS, but is inhibited by an antioxidant, N-acetyl-L-cysteine (NAC). Modification of TNFalpha production is associated with an enhancement or a repression of NF-kappaB activity by NADPH or NAC, respectively. Taken together, these results indicate that: (a) activation of the TNFalpha gene promoter contributes to the vanadate-induced TNFalpha production; (b) NF-kappaB is required for the vanadate-induced promoter activity of TNFalpha gene; (c) free radical reactions are involved in the vanadate-induced TNFalpha production and NF-kappaB activation.
Phosphatidylinositol 3-kinase (PI3-K) phosphorylates the 3-position of phosphatidylinositol to give rise to three signaling phospholipids. Binding of the pleckstrin homology (PH) domain of Akt to membrane PI(3)P's causes the translocation of Akt to the plasma membrane bringing it into contact with membrane-bound Akt kinase (PDK1 and 2), which phosphorylates and activates Akt. Akt inhibits apoptosis by phosphorylating Bad, thus promoting its binding to and blockade of the activity of the cell survival factor Bcl-x. Herein we present the synthesis and biological activity of several novel phosphatidylinositol analogues and demonstrate the ability of the carbonate group to function as a surrogate for the phosphate moiety. Due to a combination of their PI3-K and Akt inhibitory activities, the PI analogues 2, 3, and 5 proved to be good inhibitors of the growth of various cancer cell lines with IC(50) values in the 1-10 microM range. The enhanced Akt inhibitory activity of the axial hydroxymethyl-bearing analogue 5 compared to its equatorial counterpart 6 is rationalized based upon postulated differences in the H-bonding patterns of these compounds in complex with a homology modeling generated structure of the PH domain of Akt. This work represents the first attempt to examine the effects of 3-modified PI analogues on these two crucial cell signaling proteins, PI3-K and Akt, in an effort to better understand their cell growth inhibitory properties.
Multiple myeloma (MM) is an invariably fatal disease that accounts for approximately 1% to 2% of all human cancers. Surprisingly little is known about the cellular pathways contributing to growth of these tumors. Although the cytokine interleukin-6 has been suggested to be the major stimulus for myeloma cell growth, the role of a second potential growth factor, insulin-like growth factor I (IGF-I), has been less clearly defined. The IGF-I signaling cascade in 8 MM cell lines was examined. In 7 of these, the IGF-I receptor (IGF-IR) was expressed and autophosphorylated in response to ligand. Downstream of IGF-IR, insulin receptor substrate 1 was phosphorylated, leading to the activation of phosphatidylinositol-3'-kinase (PI-3K). PI-3K, in turn, regulated 2 distinct pathways. The first included Akt and Bad, leading to an inhibition of apoptosis; the second included the mitogen-activated protein kinase (MAPK), resulting in proliferation. Biologic relevance of this pathway was demonstrated because in vitro IGF-I induced both an antiapoptotic and a proliferative effect. Importantly, in vivo administration of IGF-I in SCID mice inoculated with the OPM-2 line led to approximately twice the growth rate of tumor cells as in controls. These results suggest that IGF-I activates at least 2 pathways effecting myeloma cell growth and contributes significantly to expansion of these cells in vivo. (Blood. 2000;96:2856-2861)
Tumor necrosis factor-alpha (TNF-α) is an important mediator during the inflammatory phase of wound healing. Excessive amounts of pro-inflammatory cytokines such as TNF-α are associated with inflammatory diseases including chronic wounds. Matrix metalloproteinases (MMPs) are involved in matrix re-modeling during wound healing, angiogenesis and tumor metastasis. As with pro- inflammatory cytokines, high levels of MMPs have been found in inflammatory states such as chronic wounds. In this report we relate these two phenomena. TNF-α stimulates secretion of active MMP-2, a type IV collagenase, in organ-cultured full-thickness human skin. This suggests a mechanism whereby excess inflammation affects normal wound healing.
To investigate this observation at the cellular and molecular levels, we examined TNF-α mediated activation of pro-MMP-2, induction of MT1-MMP, and the intracellular signaling pathways that regulate the proteinase in isolated human dermal fibroblasts. We found that TNF-α substantially promoted activation of pro- MMP-2 in dermal fibroblasts embedded in type-I collagen. In marked contrast, collagen or TNF-α individually had little influence on the fibroblast-mediated pro-MMP-2 activation. One well-characterized mechanism for pro- MMP-2 activation is through a membrane type matrix metalloproteinase, such as MT1-MMP. We report that TNF-α significantly induced MT1-MMP at the mRNA and protein levels when the dermal fibroblasts were grown in collagen. Although the intracellular signaling pathway regulating mt1-mmp gene expression is still obscure, both TNF-α and collagen activate the NF-κB pathway. In this report we provide three sets of evidence to support a hypothesis that activation of NF-κB is essential to induce MT1-MMP expression in fibroblasts after TNF-α exposure. First, SN50, a peptide inhibitor for NF-κB nuclear translocation, simultaneously blocked the TNF-α and collagen mediated MT1-MMP induction and pro-MMP-2 activation. Secondly, TNF-α induced IκB to breakdown in fibroblasts within the collagen lattice, a critical step leading to NF-κB activation. Lastly, a consensus binding site for p65 NF-κB (TGGAGCTTCC) was found in the 5′-flanking region of human mt1-mmp gene.
Based on these results and previous reports, we propose a model to explain TNF-α activation of MMP-2 in human skin. Activation of NF-κB signaling in fibroblasts embedded in collagen induces mt1-mmp gene expression, which subsequently activates the pro-MMP-2. The findings provide a specific mechanism whereby TNF-α may affect matrix remodeling during wound healing and other physiological and pathological processes.
Although X-linked inhibitor of apoptosis protein (Xiap) is an important intracellular suppressor of apoptosis in a variety of cell types and is present in ovary, its physiological role in follicular development remains unclear. The purpose of the present studies was to examine the modulatory role of Xiap in the proapoptotic action of tumor necrosis factor-alpha (TNFalpha) in rat granulosa cells. Granulosa cells from equine CG-primed immature rats were plated in RPMI 1640 medium containing 10% FCS and subsequently cultured in serum-free RPMI in the absence or presence of TNFalpha (20 ng/ml), the protein synthesis inhibitor cycloheximide (10 microM), and/or adenoviral Xiap sense or antisense complementary DNA. TNFalpha alone failed to induce granulosa cell death, but in the presence of cycloheximide, it markedly increased the number of apoptotic granulosa cells (as assessed by in situ terminal deoxynucleotidyl transferase-mediated deox-UTPbiotin end labeling and DNA fragmentation analysis). Western analysis indicated that TNFalpha alone increased the Xiap protein level, a response significantly reduced by adenoviral Xiap antisense expression. Down-regulation of Xiap expression by antisense complementary DNA induced granulosa cell apoptosis, which was potentiated by the cytokine. Inhibition of nuclear factor-kappaB activation by N-acetyl-cysteine and SN50 suppressed Xiap protein expression and enhanced apoptosis induced by TNFalpha. The latter phenomenon was readily attenuated by adenoviral Xiap sense expression. In conclusion, these findings suggest that Xiap is an important intracellular modulator of the TNFalpha death signaling pathway in granulosa cells. Its expression is regulated by the TNFalpha via a nuclear factor-kappaB-mediated mechanism.
Multiple myeloma (MM) remains incurable and novel treatments are urgently needed. Preclinical in vitro and in vivo evaluations were performed to assess the potential therapeutic applications of human recombinant tumor necrosis factor (TNF)-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) in MM. TRAIL/Apo2L potently induced apoptosis of MM cells from patients and the majority of MM cell lines, including cells sensitive or resistant to dexamethasone (Dex), doxorubicin (Dox), melphalan, and mitoxantrone. TRAIL/Apo2L also overcame the survival effect of interleukin 6 on MM cells and did not affect the survival of peripheral blood and bone marrow mononuclear cells and purified B cells from healthy donors. The status of the TRAIL receptors (assessed by immunoblotting and flow cytometry) could not predict TRAIL sensitivity of MM cells. The anti-MM activity of TRAIL/Apo2L was confirmed in nu/xid/bg mice xenografted with human MM cells; TRAIL (500 microg intraperitoneally daily for 14 days) was well tolerated and significantly suppressed the growth of plasmacytomas. Dox up-regulated the expression of the TRAIL receptor death receptor 5 (DR5) and synergistically enhanced the effect of TRAIL not only against MM cells sensitive to, but also against those resistant to, Dex- or Dox-induced apoptosis. Nuclear factor (NF)-kappaB inhibitors, such as SN50 (a cell-permeable inhibitor of the nuclear translocation and transcriptional activity of NF-kappaB) or the proteasome inhibitor PS-341, enhanced the proapoptotic activity of TRAIL/Apo2L against TRAIL-sensitive MM cells, whereas SN50 reversed the TRAIL resistance of ARH-77 and IM-9 MM cells. Importantly, normal B lymphocytes were not sensitized to TRAIL by either Dox, SN50, or PS-341. These preclinical studies suggest that TRAIL/Apo2L can overcome conventional drug resistance and provide the basis for clinical trials of TRAIL-based treatment regimens to improve outcome in patients with MM. (Blood. 2001;98:795-804)
In this study we demonstrate that tumor necrosis factor alpha (TNFalpha) triggers only modest proliferation, as well as p44/p42 mitogen-activated protein kinase (MAPK) and NF-kappaB activation, in MM.1S multiple myeloma (MM) cells. TNFalpha also activates NF-kappaB and markedly upregulates (fivefold) secretion of interleukin-6 (IL-6), a myeloma growth and survival factor, in bone marrow stromal cells (BMSCs). TNFalpha in both a dose and time dependent fashion induced expression of CD11a (LFA-1), CD54 (intercellular adhesion molecule-1, ICAM-1), CD106 (vascular cell adhesion molecule-1, VCAM-1), CD49d (very late activating antigen-4, VLA-4), and/or MUC-1 on MM cell lines; as well as CD106 (VCAM-1) and CD54 (ICAM-1) expression on BMSCs. This resulted in increased (2-4-fold) per cent specific binding of MM cells to BMSCs, with related IL-6 secretion. Importantly, the proteasome inhibitor PS-341 abrogated TNFalpha-induced NF-kappaB activation, induction of ICAM-1 or VCAM-1, and increased adhesion of MM cells to BMSCs. Agents which act to inhibit TNFalpha may therefore abrogate the paracrine growth and survival advantage conferred by MM cell adhesion in the BM microenvironment.
Previous studies demonstrate that interleukin-6 (IL-6) mediates growth and survival in human multiple myeloma (MM) cells via the MEK/MAPK and JAK/STAT signaling pathways, respectively. IL-6 also confers protection against Dexamethasone (Dex)-induced apoptosis via activation of protein tyrosine phosphatase (SHP2). In the current study, we characterized IL-6 triggered phophatidylinositol-3 kinase/Akt kinase (PI3-K/Akt) signaling in MM cells. IL-6 induces Akt/PKB phosphorylation in a time and dose dependent manner in MM.1S MM cells. IL-6 also induced phosphorylation of downstream targets of Akt, including Bad, GSK-3beta, and FKHR, confirming Akt activation. Inhibition of Akt activation by the PI3-K inhibitor LY294002 partially blocked IL-6 triggered MEK/MAPK activation and proliferation in MM.1S cells, suggesting cross-talk between PI3-K and MEK signaling. We demonstrate that Dex-induced apoptosis in MM.1S cells is mediated by downstream activation of caspase-9, with resultant caspase-3 cleavage; and conversely, that IL-6 triggers activation of PI3-K and its association with SHP2, inactivates caspase-9, and protects against Dex-induced apoptosis. LY294002 completely abrogates this signaling cascade, further confirming the importance of PI3-K/Akt signaling in conferring the protective effect of IL-6 against Dex-induced apoptosis. Finally, we show that IL-6 triggered PI3-K/Akt signaling in MM.1S cells inactivates forkhead transcriptional factor (FKHR), with related G1/S phase transition, whereas LY294002 blocks this signaling, resulting in upregulation of p27(KIP1) and G1 growth arrest. Our data therefore suggest that PI3-K/Akt signaling mediates growth, survival, and cell cycle regulatory effects of IL-6 in MM.
Leukocyte adhesion to the diabetic retinal vasculature results in blood-retinal barrier breakdown, capillary nonperfusion, and endothelial cell injury and death. Intercellular adhesion molecule-1 (ICAM-1) and the leukocyte integrin CD18 are required for these processes. Diabetes was induced in Long Evans rats, resulting in a two- to threefold increase in retinal leukocyte adhesion. Following one week of diabetes, neutrophil CD11a, CD11b, and CD18 expression was increased significantly, as were retinal ICAM-1 levels. Animals were treated with aspirin, a cyclooxygenase 2 (COX-2) inhibitor (meloxicam), or a soluble tumor necrosis factor alpha (TNF-alpha) receptor/Fc construct (TNFR-Fc, etanercept). High-dose aspirin, etanercept, and high-dose meloxicam each reduced leukocyte adhesion and suppressed blood-retinal barrier breakdown. High-dose aspirin also reduced the expression of CD11a, CD11b, and CD18, whereas meloxicam and etanercept did not. High-dose aspirin, etanercept, and high-dose meloxicam each reduced retinal ICAM-1 expression. Aspirin and meloxicam both lowered retinal TNF-alpha levels. Notably, aspirin, meloxicam, and etanercept did not change retinal vascular endothelial growth factor levels. High-dose aspirin, etanercept and high-dose meloxicam, each suppressed the retinal expression of eNOS and the DNA-binding capacity of retinal nuclear factor-kappaB. High-dose aspirin also suppressed Erk kinase activity, which is involved in CD18 up-regulation. Taken together, these data identify COX-2 and TNF-alpha as operative in the early signature pathologies of diabetic retinopathy, a newly recognized inflammatory disease.
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL, Apo2 ligand) effectively kills multiple myeloma (MM) cells in vitro irrespective of refractoriness to dexamethasone and chemotherapy. Because clinical trials with this anticancer agent are expected shortly, we investigated the signaling pathway of TRAIL-induced apoptosis in MM. We detected rapid cleavage of caspases-8, -9, -3, and -6, as well as the caspase substrates poly(ADP-ribose) polymerase (PARP) and DNA fragmentation factor-45 (DFF45), but not caspase-10, upon TRAIL treatment in sensitive MM cells, pointing to caspase-8 as the apical caspase of TRAIL signaling in MM cells. These phenomena were not observed or were significantly delayed in TRAIL-resistant MM cells, suggesting that resistance may arise from inhibition at the level of caspase-8 activation. Higher levels of expression for various apoptosis inhibitors, including FLICE-inhibitory protein (FLIP), and lower procaspase-8 levels were present in TRAIL-resistant cells and sensitivity was restored by the protein synthesis inhibitor cycloheximide (CHX) and the protein kinase C (PKC) inhibitor bisindolylmaleimide (BIM), which both lowered FLIP and cellular inhibitor of apoptosis protein-2 (cIAP-2) protein levels. Forced expression of procaspase-8 or FLIP antisense oligonucleotides also sensitized TRAIL-resistant cells to TRAIL. Moreover, the cell permeable nuclear factor (NF)-kappaB inhibitor SN50, which sensitizes TRAIL-resistant cells to TRAIL, also inhibited cIAP2 protein expression. Finally, CHX, BIM, and SN50 facilitated the cleavage and activation of procaspase-8 in TRAIL-resistant cells, confirming that inhibition of TRAIL-induced apoptosis occurs at this level and that these agents sensitize MM cells by relieving this block. Our data set a framework for the clinical use of approaches that sensitize MM cells to TRAIL by agents that inhibit FLIP and cIAP-2 expression or augment caspase-8 activity.
The transcription factor nuclear factor-kappaB (NF-kappaB) confers significant survival potential in a variety of tumors. Several established or novel anti-multiple myeloma (anti-MM) agents, such as dexamethasone, thalidomide, and proteasome inhibitors (PS-341), inhibit NF-kappaB activity as part of their diverse actions. However, studies to date have not delineated the effects of specific inhibition of NF-kappaB activity in MM. We therefore investigated the effect of SN50, a cell-permeable specific inhibitor of NF-kappaB nuclear translocation and activity, on MM cells. SN50 induced apoptosis in MM cell lines and patient cells; down-regulated expression of Bcl-2, A1, X-chromosome-linked inhibitor-of-apoptosis protein (XIAP), cellular inhibitor-of-apoptosis protein 1 (cIAP-1), cIAP-2, and survivin; up-regulated Bax; increased mitochondrial cytochrome c release into the cytoplasm; and activated caspase-9 and caspase-3, but not caspase-8. We have previously demonstrated that tumor necrosis factor-alpha (TNF-alpha) is present locally in the bone marrow microenvironment and induces NF-kappaB-dependent up-regulation of adhesion molecules on both MM cells and bone marrow stromal cells, with resultant increased adhesion. In this study, TNF-alpha alone induced NF-kappaB nuclear translocation, cIAP-1 and cIAP-2 up-regulation, and MM cell proliferation; in contrast, SN50 pretreatment sensitized MM cells to TNF-alpha-induced apoptosis and cleavage of caspase-8 and caspase-3, similar to our previous finding of SN50-induced sensitization to apoptosis induced by the TNF-alpha family member TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L. Moreover, SN50 inhibited TNF-alpha-induced expression of another NF-kappaB target gene, intercellular adhesion molecule-1. Although the p38 inhibitor PD169316 did not directly kill MM cells, it potentiated the apoptotic effect of SN50, suggesting an interaction between the p38 and NF-kappaB pathways. Our results therefore demonstrate that NF-kappaB activity in MM cells promotes tumor-cell survival and protects against apoptotic stimuli. These studies provide the framework for targeting NF-kappaB activity in novel biologically based therapies for MM.
Thalidomide (Thal) achieves responses even in the setting of refractory multiple myeloma (MM). Although increased angiogenesis in MM bone marrow and the antiangiogenic effect of Thal formed the empiric basis for its use in MM, we have shown that Thal and its immunomodulatory analogs (IMiDs) directly induce apoptosis or growth arrest of MM cells, alter adhesion of MM cells to bone marrow stromal cells, inhibit the production of cytokines (interleukin-6 and vascular endothelial growth factor) in bone marrow, and stimulate natural killer cell anti-MM immunity. In the present study, we demonstrate that the IMiDs trigger activation of caspase-8, enhance MM cell sensitivity to Fas-induced apoptosis, and down-regulate nuclear factor (NF)-kappa B activity as well as expression of cellular inhibitor of apoptosis protein-2 and FLICE inhibitory protein. IMiDs also block the stimulatory effect of insulinlike growth factor-1 on NF-kappa B activity and potentiate the activity of TNF-related apoptosis-inducing ligand (TRAIL/Apo2L), dexamethasone, and proteasome inhibitor (PS-341) therapy. These studies both delineate the mechanism of action of IMiDs against MM cells in vitro and form the basis for clinical trials of these agents, alone and coupled with conventional and other novel therapies, to improve outcome in MM.
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